NANC inhibitory neurotransmission in mouse isolated stomach: involvement of nitric oxide, ATP and vasoactive intestinal polypeptide

Abstract

1. The neurotransmitters involved in NANC relaxation and their possible interactions were investigated in mouse isolated stomach, recording the motor responses as changes of endoluminal pressure from whole organ. 2. Field stimulation produced tetrodotoxin-sensitive, frequency-dependent, biphasic responses: rapid transient relaxation followed by a delayed inhibitory component. 3. The inhibitor of the synthesis of nitric oxide (NO), l-NAME, abolished the rapid relaxation and significantly reduced the slow relaxation. Apamin, blocker of Ca2+-dependent K+ channels, or ADPbetaS, which desensitises P2y purinoceptors, reduced the slow relaxation to 2-8 Hz, without affecting that to 16-32 Hz or the fast relaxation. alpha-Chymotrypsin or vasoactive intestinal polypeptide 6-28 (VIP6-28), antagonist of VIP receptors, failed to affect the fast component or the delayed relaxation to 2-4 Hz, but antagonised the slow component to 8-32 Hz. 4. Relaxation to sodium nitroprusside was not affected by l-NAME, apamin or ADPbetaS, but was reduced by alpha-chymotrypsin or VIP6-28. Relaxation to VIP was abolished by alpha-chymotrypsin, antagonised by VIP6-28, but was not affected by l-NAME, apamin or ADPbetaS. Relaxation to ATP was abolished by apamin, antagonised by ADPbetaS, but was not affected by l-NAME or alpha-chymotrypsin. 5. The present results suggest that NO is responsible for the rapid relaxation and partly for the slow relaxation. ATP is involved in the slow relaxation evoked by low frequencies of stimulation. VIP is responsible for the slow relaxation evoked by high frequencies of stimulation. The different neurotransmitters appear to work in parallel, although NO could serve also as a neuromodulator that facilitates release of VIP.

Figure 1

Original tracings illustrating the effect of L-NAME on the EFS-evoked NANC relaxations. EFS (0.5 ms, supramaximal voltage, for 5 s, 2–32 Hz) evoked biphasic responses, consisting of a fast relaxation followed by a slow relaxation. L-NAME (300 μM) abolished the early fast component and reduced the slow relaxation. Arrows indicate EFS.

Figure 2

Effects of apamin on the amplitude of the two components of NANC relaxation evoked by different stimulation frequencies. Apamin (0.1 μM) did not modify the fast relaxation, but it significantly reduced the slow relaxation to 2–8 Hz, without affecting that to higher frequencies of stimulation. All values are mean±s.e.m., n=4. ^*P<0.05 when compared to the respective control conditions (using Student's t-test).

Figure 3

Effects of the desensitisation of the P_2y purinergic receptors with ADPβs on the amplitude of the two components of NANC relaxation evoked by different stimulation frequencies. ADPβs (10 μM for 30 min) did not modify the fast relaxation, but it significantly reduced the slow relaxation to 2–8 Hz, without affecting that to higher frequency of stimulation. The subsequent addition of L-NAME (300 μM) abolished the fast relaxation and the slow relaxation to 2–8 Hz, and significantly reduced the second slow component to 16–32 Hz. All values are mean±s.e.m., n=4. ^*P<0.05 when compared to the respective control conditions (using ANOVA followed by Bonferroni's t-test).

Figure 4

Effects of α-chymotrypsin on the amplitude of the two components of NANC relaxation evoked by different stimulation frequencies. α-chymotrypsin (10 U ml⁻¹) did not modify the fast relaxation and the slow relaxation to low frequencies, but it significantly reduced that to high frequencies of stimulation. All values are mean±s.e.m., n=6. ^*P<0.05 when compared to the respective control conditions (using Student's t-test).

Figure 5

Effects of VIP6–28, a selective antagonist of VIP receptors, on the amplitude of the two components of NANC relaxation evoked by different stimulation frequencies. VIP6–28 (10 μM) did not modify the fast relaxation and the slow relaxation to low frequencies, but it significantly reduced that to high frequencies of stimulation. The subsequent addition of L-NAME (300 μM) abolished the fast relaxations and the slow relaxations to 16–32 Hz, and significantly reduced the second slow component to 2–8 Hz. All values are mean±s.e.m., n=4. ^*P<0.05 when compared to the respective control conditions (using ANOVA followed by Bonferroni's t-test).

Figure 6

Concentration–response curves for SNP-induced relaxation in mouse gastric preparations before and after different pharmacological treatment. Apamin (0.1 μM, n=4), ADPβs (10 μM, n=5) and L-NAME (300 μM, n=5) did not alter the responses to SNP. α-Chymotrypsin (αCT) (10 U ml⁻¹, n=5) and VIP6–28 (10 μM, n=5) reduced the effectiveness of SNP. All values are mean±s.e.m., and are reported as a percentage of the maximum effect induced by 10 μM SNP. ^*P<0.05 vs control (using paired Student's t-test).

Figure 7

Concentration–response curves for VIP-induced relaxation in mouse gastric preparations before and after different pharmacological treatment. Apamin (0.1 μM, n=4), ADPβs (10 μM, n=5) and L-NAME (300 μM, n=5) did not alter the responses to VIP, whereas α-chymotrypsin (αCT) (10 u ml⁻¹, n=5) abolished and VIP6–28 (10 μM, n=5) antagonised the response to VIP. All values are mean±s.e.m., and are reported as a percentage of the maximum effect induced by 0.3 μM VIP. ^*P<0.05 vs control (using Student's t-test).

Figure 8

Concentration–response curves for ATP-induced relaxation in mouse gastric preparations before and after different pharmacological treatment. The response to ATP was abolished by apamin (0.1 μM, n=4), antagonised by ADPβs (10 μM, n=5) and was not altered by L-NAME (300 μM, n=5) or by α-chymotrypsin (αCT) (10 U ml⁻¹, n=5). All values are mean±s.e.m., and are reported as a percentage of the maximum effect induced by 1 μM ATP. ^*P<0.05 vs control (using paired Student's t-test).