The effect of epibatidine on spontaneous and evoked neurotransmitter release in the mouse and guinea pig isolated vas deferens

Abstract

Background and purpose: Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species.

Experimental approach: The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques.

Key results: In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 microM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or alpha,beta-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, alpha,beta-methyleneATP and cyclopentolate. In guinea pig vasa deferentia, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes.

Conclusion and implications: The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.

Figure 1

The effect of slow and rapid application of epibatidine (100 nM) on neurotransmitter release in the mouse vas deferens. (a) A typical trace of the membrane potential following slow, bath application of epibatidine (100 nM). EJPs were evoked at a frequency of 0.33 Hz. In most experiments, there was no clear effect of epibatidine on EJP amplitude or SEJP frequency. (b) In two experiments, a weak potentiation of EJPs and a small increase in SEJP frequency was observed following addition of epibatidine. (c) A typical electrophysiological trace showing that the rapid application of 100 nM epibatidine had no significant effect on EJP amplitude but clearly increased SEJP frequency. Portions of the trace before and following the addition of epibatidine are expanded to show SEJPs more clearly. The apparent depolarisation of the RMP shown in (c) was not consistently observed. (d) A typical trace showing that following incubation with hexamethonium (100 μM) for 60 min, the increase in SEJP induced by rapid application of epibatidine is abolished.

Figure 2

The effect of bath application of epibatidine (100 nM) on EJP amplitude in the guinea pig vas deferens. (a) Typical traces showing membrane potential of the smooth muscle cells following neuronal stimulation at 0.33 Hz. (i) After addition of epibatidine, there was a transient large potentiation in EJP amplitude followed by a more sustained, smaller potentiation of EJP amplitude. (ii) In the presence of hexamethonium (100 μM) for 60 min, the potentiating effects of epibatidine were blocked. (b) Graph showing the effect of epibatidine alone and in the presence of hexamethonium on normalised EJP amplitude. Results shown are means±s.e.m. from 11 (epibatidine alone) or 13 (epibatidine+hexamethonium) experiments.

Figure 3

The effect of epibatidine (100 nM) on neurogenic contraction in the mouse vas deferens. (a) A trace showing the effect of epibatidine (100 nM) on neurogenic contraction in the mouse vas deferens. In 50% of the preparations, a transient contraction was observed following epibatidine addition. Neurogenic contractions, evoked every 30 s by a train of five stimuli at 10 Hz (marked by the dots below the trace) were potentiated following epibatidine addition. (b) Following the addition of prazosin (100 nM) for 40 min, the amplitude of neurogenic contractions was still potentiated by epibatidine. On both traces, a movement artefact is visible when epibatidine is added.

Figure 4

Epibatidine-induced contraction of mouse vas deferens. (a) Typical traces showing a transient contraction following the addition of epibatidine (1 μM), either (i) without other drugs or (ii) following pre-treatment with TTX 300 nM for 40 min, (iii) α,β-MeATP 1 μM for 1 h or (iv) prazosin 100 nM for 40 min. Pretreatment with (v) both α,β-MeATP and prazosin or (vi) α,β-MeATP, prazosin and cyclopentolate 1 μM for 1 h, led to further diminution of the epibatidine-induced contraction. (b) Bar chart showing the amplitude of epibatidine-induced contractions in the presence of various drugs. Each drug significantly reduced the amplitude of contraction in comparison with control experiments (P<0.05). In the presence of cyclopentolate, α,β-MeATP and prazosin, the amplitude of contraction was below the level of detection. Results shown are means±s.e.m. from 6 to 11 experiments in each group.