Post‐ and prejunctional consequences of ecto‐ATPase inhibition: electrical and contractile studies in guinea‐pig vas deferens

Abstract

At sites of purinergic neurotransmission, synaptic ecto-ATPase is believed to limit the actions of ATP following its neural release. However, details of the modulation by this enzyme of the ATP-mediated conductance change and the possible mechanisms mediating this modulation remain unelucidated. We have addressed these issues by studying the effect of ARL 67156, a selective ecto-ATPase inhibitor, on ATP-mediated electrical and contractile activity in the sympathetically innervated guinea-pig vas deferens. ARL 67156 at 100 μM significantly potentiated the amplitude of spontaneous excitatory junction potentials (SEJPs) by 81.1% (P < 0.01) and prolonged their time courses (rise time by 49.7%, decay time constant by 38.2%; P < 0.01). Moreover, the frequency of occurrence of SEJPs was strikingly increased (from 0.28 ± 0.13 to 0.90 ± 0.26 Hz; P < 0.01), indicating an additional, primarily presynaptic, effect of ecto-ATPase inhibition. The frequency of occurrence of discrete events (DEs), which represent nerve stimulation-evoked quantal release of neurotransmitter, was also increased (∼6-fold; P < 0.01), along with the appearance of DEs at previously 'silent' latencies. Purinergic contractions of the vas deferens were potentiated significantly (P < 0.01) by ARL 67156; these potentiated contractions were suppressed by the A1 agonist adenosine (P < 0.01) but left unaffected by the A1 antagonist 8-phenyltheophylline (8-PT). Our results indicate (i) that ecto-ATPase activity, in addition to modulating the ATP-mediated postjunctional conductance change, may regulate transmitter release prejunctionally under physiological conditions, and (ii) that the prejunctional regulation may be mediated primarily via presynaptic P2X, rather than A1, receptors.

Targeted Deletion of Ectonucleoside Triphosphate Diphosphohydrolase 1/CD39 Leads to Desensitization of Pre- and Postsynaptic Purinergic P2 Receptors

“…At synapses where transmitter lifetime is controlled by enzymatic hydrolysis, inhibition of inactivating enzymes can profoundly affect changes in membrane conductance elicited by neurotransmitters. Indeed, inhibition of ecto-ATPase by the selective inhibitor ARL67156 potentiates excitatory junction potentials, ATP overflow, and development of spontaneous excitatory junction potentials in guinea pig vas deferens Ghildyal et al, 2006). Therefore, at the sympathetic neuroeffector junction, ectoATPase modulates the prejunctional release of ATP as well as its postjunctional actions.…”

Cellular function and molecular structure of ecto-nucleotidases

“…There are multiple examples showing that application of inhibitors such as the nucleotide analogue ARL 67156 potentiate nucleotide-mediated neurotransmission [170][171][172][173]. Deletion of NTPDase1 in mice revealed a key role of this enzyme in the prevention of thrombosis [41,42], in purine signaling in angiogenesis [174], vascular permeability [175,176], vascular relaxation [49], in the control of macrophage function [177], or also in promoting tumor growth [178].…”

Loss of purinergic vascular regulation in the colon during colitis is associated with upregulation of CD39

“…The RNA was reprecipitated using LiCl precipitation solution (Ambion). Subsequently, cDNA was reverse transcribed from 1 g of total RNA using SuperScript III (Invitrogen) and oligo(dT) [12][13][14][15][16][17][18] primers (Invitrogen). Real-time PCR was performed using the primers listed in Table 2 with a Roche Lightcycler and the Quantitech SYBR Green PCR kit (Qiagen).…”