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 mum 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 ( approximately 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.

Figure 1. Effect of ARL 67156 on junction potentials

A, amplitude histogram of SEJPs. Note, in the presence of 100 μm ARL 67156, the occurrence of high-amplitude SEJPs and the reduction in numbers of low-amplitude events. B, rise time (a) and decay (b) of representative SEJPs before (Control) and after exposure to ARL 67156. The SEJPs analysed are shown in the inset. C, comparison of time courses of averaged traces of 5 EJPs recorded in the same cell in the absence (Control) and presence of 100 μm ARL 67156. Inset shows the rising phases of the traces after normalization.

Figure 2. Effect of ARL 67156 on SEJP frequency

A, SEJPs recorded from two different cells before and after addition of 100 μm ARL 67156. Note the increase in frequency of occurrence of SEJPs. B, SEJP frequency before (control) and after ARL 67156 treatment measured during an intracellular electrical recording from a single cell. SEJP occurrence has been indicated as the number of events per 10 s. While the average frequency of SEJP occurrence in control was 0.41 Hz, in ARL 67156 treated cells it rose to 1.08 Hz. C, inter-event intervals of SEJPs recorded from a single cell before and after ARL 67156 application. Mean interevent interval for control SEJPs was 3.2 s and for those in the presence of ARL 67156 was 1.5 s (Kolmogorov-Smirnov test; P < 0.01).

Figure 3. Effect of ARL 67156 on Des

A, rising phases of EJPs overlaid with the traces of their first time differential (DEs). Note, the increase in the number of DEs in the presence of ARL 67156. B, frequency of DE occurrence in a train of 50 EJPs recorded in a single cell. The DE frequency in control EJPs was 0.3 DEs per EJP, and 1.5 DEs per EJP after exposure to 100 μm ARL 67156. C, plot of individual DE amplitudes against their latencies. While the mean DE amplitude remained unaffected, note the emergence of DEs with latencies beyond 80 ms (to the right of the vertical line) in the presence of ARL 67156. D, amplitude distribution of the rate of rise of SEJPs. SEJP rate of rise remained unchanged in the presence of ARL 67156.

Figure 4. Effect of ARL 67156 on phasic contractions in the presence of adenosine and 8-PT

A and B, traces showing the effect on phasic contractions of a single tissue of the A1 agonist adenosine (Ade) (A) and the A1 antagonist 8-phenyltheophylline (8-PT) (B). Duration of stimulation is indicated by the bar under the traces. Aa and Ba, control phasic contractions (in the presence of prazosin, P and yohimbine, Y); Ab and Bb, phasic contractions in the presence of Ade and 8-PT, respectively. Ac and Bc, effect of ARL 67156 (ARL) on phasic contractions in the presence of Ade and 8-PT, respectively. Note, while Ade reduced the control contraction (Ab), 8-PT had no effect (Bb). ARL significantly enhanced the phasic contractions, both in the presence of Ade and in the presence of 8-PT. C and D, bar chart of the effect of ARL on phasic contractions in the presence of Ade and 8-PT, respectively (pooled data). Note, while exogenously added Ade inhibited phasic contractions both alone (by ∼27%) and in the presence of 100 μm ARL (by ∼50%) (P < 0.01), 8-PT did not affect the phasic contractions significantly (NS) either alone or in the presence of 100 μm ARL (P > 0.05).