Peter Sneddon

Enhancement of sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens by the novel ecto-ATPase inhibitor ARL 67156.

1 Field stimulation of the sympathetic nerves of the guinea‐pig isolated vas deferens with trains of pulses for 20 s at 1–8 Hz produced characteristic biphasic contractions. The effect of the novel ecto‐ATPase inhibitor, 6‐N,N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156, formerly known as FPL 67156), on the magnitude of the initial, predominantly purinergic peak of this response was studied in order to determine the influence of enzymatic degradation of adenosine 5′‐triphosphate (ATP) on its action as a neurotransmitter. 2 The peak magnitude of the response to nerve stimulation was significantly increased in a concentration‐dependent manner by ARL 67156 (5–100 μm) and the size of the neurogenic response at 4 Hz was approximately doubled in the presence of ARL 67156 (100 μm). 3 ARL 67156 (100 μm) has a rapid onset of action. The enhancing effect on neurogenic contractions was maximal after 10 min, was well maintained for at least 30 min and was rapidly reversed, with responses returning to control levels 10 min after washout. 4 The neurogenic contraction in the presence of prazosin (0.1 μm) was purely purinergic, as it was abolished by the P2‐purinoceptor antagonist, PPADS (100 μm). ARL 67156 (100 μm) produced a similar degree of enhancement of neurogenic responses in the absence and presence of prazosin, supporting the view that the enhancing effects of ARL 67156 on neurogenic contractions result from potentiation of the action of ATP. 5 Exogenous ATP and α,β‐methyleneATP produced rapid transient contractions. Responses to ATP were increased in magnitude and duration in the presence of ARL 67156 (100 μm), whereas those to the stable analogue, α,β‐methyleneATP were not significantly affected. 6 Contractions to exogenous noradrenaline (10 μm) and KCl (40 mM) were significantly enhanced by ARL 67156 (100 μm), but this potentiation was abolished by PPADS (100 μm). Therefore, this effect of the ecto‐ATPase inhibitor may be due to a build up of endogenous ATP, increasing the sensitivity of the smooth muscle to other agonists. 7 It is concluded that ARL 67156 potentiates the action of ATP, and that when ATP acts as a neurotransmitter its postjunctional actions are greatly attenuated by enzymatic degradation.

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.

Evidence for ATP as a cotransmitter in dog mesenteric artery

Contractile responses of the dog mesenteric artery were obtained (after removal of endothelium) to transmural stimulation of the perivascular nerves and to exogenous application of ATP, noradrenaline, dopamine, 5-hydroxy-tryptamine and high potassium solution. The alpha-adrenoceptor antagonists prazosin and phentolamine preferentially reduced the response to noradrenaline and the secondary phase of the biphasic contractile response to nerve stimulation, whilst the addition of alpha, beta-methylene-ATP, which selectively desensitizes P2-purinoceptors, reduced only the contractions to ATP and the portion of the nerve-mediated response which was resistant to the alpha-adrenoceptor antagonists. The responses to nerve stimulation were reduced by the selective P1-purinoceptor agonist 2-chloroadenosine, and its effect was reversed by the P1-purinoceptor antagonist 8-phenyltheophylline. These results suggest that in dog mesenteric artery part of the response to sympathetic nerve stimulation is mediated by ATP acting on P1-purinoceptors on the arterial smooth muscle, and that P1-purinoceptors on the sympathetic nerve terminal can inhibit release of the neurotransmitters.

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

The site(s) at which P2‐receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2‐receptor antagonists and the extracellular ATPase inhibitor 6‐N,N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156). Suramin (1 μM–1 mM) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi‐effective concentrations of α,β‐methyleneATP (α,β‐meATP) (5 μM), 2‐methylthioATP (2‐meSATP) (100 μM) and adenosine 5′‐triphosphate (ATP) (1 mM) in a concentration‐dependent manner. Responses to α,β‐meATP and 2‐meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS. Contractions evoked by uridine 5′‐triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM). Desensitization of the P2X1‐receptor by α,β‐meATP abolished contractions evoked by 2‐meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control. Responses to α,β‐meATP (5 μM) and 2‐meSATP (100 μM) were abolished when tissues were bathed in nominally calcium‐free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β‐meATP (5 μM), 2‐meSATP (100 μM) and ATP (1 mM) in a concentration‐dependent manner. These results suggest that two populations of P2‐receptors are present in the rat tail artery; ligand‐gated P2X1‐receptors and G‐protein‐coupled P2Y‐receptors.

Chapter 2 Modulation of purinergic neurotransmission

Publisher Summary This chapter highlights some recent developments in the understanding of ATP as a cotransmitter. Four main topics of purinergic research are emphasized in the chapter: the storage and release of ATP and its regulation, the structure and classification of P2-receptor subtypes, the postjunctional effector mechanisms by which ATP mediates its neurotransmitter actions, and the mechanism of inactivation of the neurotransmitter actions of ATP by ATPases. Recent studies indicate that there are more than one population of storage vesicles in the nerves, as the release of various cotransmitters varies over time and can be differentially modulated by drugs. The subclassification of P2 receptors has advanced in the past few years because of the use of molecular biology methods allowing the cloning and expression of 14 different subclasses of P2 receptors, seven P2X, and seven P2Y. Determination of the functional significance of various receptor subtypes would be helped by the development of selective agonists and antagonists.

Suramin inhibits excitatory junction potentials in guinea-pig isolated vas deferens.

1 . Intracellular microelectrode recording techniques were used to investigate the action of the putative P2‐purinoceptor antagonist, suramin, on sympathetic neurotransmission in the guinea‐pig isolated vas deferens. 2 . The resting membrane potential of the control cells was 67.4 ± 0.7 mV (n = 48). Field stimulation of the sympathetic nerves innervating the vas deferens produced excitatory junction potentials (e.j.ps) which reached a mean magnitude of 8.5 ± 0.8 mV (n = 23) when fully facilitated at a stimulation frequency of 0.5 Hz. 3 . Introduction of suramin 1–100 μm produced no change in the resting membrane potential of the smooth muscle cells, but gradually reduced e.j.p. magnitude. Suramin, 20 μm, reduced the mean magnitude of the fully facilitated e.j.ps to 1.4 ± 0.3mV (n = 18). 4 . After suramin‐induced inhibition of e.j.ps, nerve stimulation at 1–8 Hz resulted in summation of e.j.ps to a subthreshold level. Subsequent introduction of the α‐adrenoceptor antagonists, prazosin or phentolamine (1 μm) did not reduce the magnitude of the summated e.j.ps. 5 . The results support the proposal that e.j.ps in vas deferens are mediated by adenosine 5′‐triphosphate, and not by noradrenaline, and confirm that suramin can antagonize responses mediated via P2‐purinoceptors.

The interaction of diadenosine polyphosphates with P2X-receptors in the guinea-pig isolated vas deferens

The site(s) at which diadenosine 5′,5′′′‐P1, P4‐tetraphosphate (AP4A) and diadenosine 5′, 5′′′‐P1, P5‐pentaphosphate (AP5A) act to evoke contraction of the guinea‐pig isolated vas deferens was studied by use of a series of P2‐receptor antagonists and the ecto‐ATPase inhibitor 6‐N, N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156). Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (300 nM–30 μM), suramin (3–100 μM) and pyridoxal‐5′‐phosphate (P‐5‐P) (3–1000 μM) inhibited contractions evoked by equi‐effective concentrations of AP5A (3 μM), AP4A (30 μM) and α,β‐methyleneATP (α,β‐meATP) (1 μM), in a concentration‐dependent manner and abolished them at the highest concentrations used. PPADS was more potent than suramin, which in turn was more potent than P‐5‐P. PPADS inhibited AP5A, AP4A and α,β‐meATP with similar IC50 values. No significant difference was found between IC50 values for suramin against α,β‐meATP and AP5A or α,β‐meATP and AP4A, but suramin was more than 2.5 times more potent against AP4A than AP5A. P‐5‐P showed the same pattern of antagonism. Desensitization of the P2X1‐receptor by α,β‐meATP abolished contractions evoked by AP5A (3 μM) and AP4A (30 μM), but had no effect on those elicited by noradrenaline (100 μM). ARL 67156 (100 μM) reversibly potentiated contractions evoked by AP4A (30 μM) by 61%, but caused a small, significant decrease in the mean response to AP5A (3 μM). It is concluded that AP4A and AP5A act at the P2X1‐receptor, or a site similar to the P2X1‐receptor, to evoke contraction of the guinea‐pig isolated vas deferens. Furthermore, the potency of AP4A, but not AP5A, appears to be inhibited by an ecto‐enzyme which is sensitive to ARL 67156.

Characterization of the ATPase released during sympathetic nerve stimulation of the guinea‐pig isolated vas deferens

The release of ATPase activity evoked by electrical field stimulation (EFS) (8 Hz, 25 s) was investigated in several tissues in which adenosine 5′‐triphosphate (ATP) acts as a neurotransmitter. Superfusate collected during EFS of sympathetic nerves of the guinea‐pig, rat and mouse isolated vas deferens and parasympathetic nerves of the guinea‐pig isolated urinary bladder contained ATPase activity. ATP breakdown was fastest in superfusate collected from the guinea‐pig isolated vas deferens. However, EFS of the enteric nerves of the guinea‐pig isolated taenia coli did not release any detectable ATPase. The ATPase released from the guinea‐pig isolated vas deferens metabolized ATP at similar rates at incubation temperatures of 37°C and 20°C. Lineweaver–Burke analysis of the initial rates of ATP hydrolysis gave a KM of 39 μM and a Vmax of 1039 pmol ATP metabolized min−1 ml−1 superfusate. 6‐N,N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156), pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) and pyridoxal‐5′‐phosphate (P‐5‐P) all inhibited the ATPase activity in a concentration‐dependent manner with a potency order of ARL 67156=PPADS>P‐5‐P. In conclusion, EFS of several tissues in which ATP is a neurotransmitter causes the release of an ATPase and activity is greatest in the guinea‐pig vas deferens. The enzyme has pharmacological and kinetic characteristics that are similar to ectonucleoside triphosphate diphosphohydrolases.

Release of soluble nucleotidases: a novel mechanism for neurotransmitter inactivation?

The support of the NIH and American Heart Foundation to Prof. D. P. Westfall, Astra Charnwood and the Wellcome Trust (C. K., P. S.), the Caledonian Research Foundation (C. K.) and Carnegie Trust (P. S.) is gratefully acknowledged. We also thank the Scottish Hospital Endowments Research Trust for refurbishing the laboratories in which some of these experiments were performed.

Electrophysiology of autonomic neuromuscular transmission involving ATP

Electrophysiological investigations of autonomic neuromuscular transmission have provided great insights into the role of ATP as a neurotransmitter. Burnstock and Holman made the first recordings of excitatory junction potentials (e.j.p.s) produced by sympathetic nerves innervating the smooth muscle of the guinea-pig vas deferens. This led to the identification of ATP as the mediator of e.j.p.s in this tissue, where ATP acts as a cotransmitter with noradrenaline. The e.j.p.s are mediated solely by ATP acting on P2X(1) receptors leading to action potentials and a rapid phasic contraction, whilst noradrenaline mediates a slower, tonic contraction which is not dependent on membrane depolarisation. Subsequent electrophysiological studies of the autonomic innervation of smooth muscles of the urogenital, gastrointestinal and cardiovascular systems have revealed a similar pattern of response, where ATP mediates a fast electrical and mechanical response, whilst another transmitter such as noradrenaline, acetylcholine, nitric oxide or a peptide mediates a slower response. The modulation of junction potentials by a variety of pre-junctional receptors and the mechanism of inactivation of ATP as a neurotransmitter will also be described.