Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

Abstract

Background and purpose: To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon.

Experimental approach: Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors.

Key results: Strips developed weak spontaneous rhythmic contractions (3.67+/-0.49 g, 2.54+/-0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 microM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1-100 microM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by N(G)-nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 microM) or the P2Y(1) receptor antagonist MRS 2179 (10 microM) and were unaffected by the P2X antagonist NF279 (10 microM) or alpha-chymotrypsin (10 U mL(-1)). Amplitude of on- and off-contractions was reduced by atropine (1 microM) and the selective NK(2) receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH(2) (1 microM). MRS 2179 reduced the amplitude of EFS on- and off-contractions without altering direct muscular contractions induced by ACh (1 nM-1 mM) or substance P (1 nM-10 microM).

Conclusions and implications: Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y(1) receptors through apamin-sensitive K(+) channels. EFS-induced on- and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK(2) receptors. Prejunctional P2Y(1) receptors might modulate the activity of excitatory EMNs. P2Y(1) and NK(2) receptors might be therapeutic targets for colonic motor disorders.

Figure 1

Experimental recordings and summary diagrams showing (a) the effect of the neural blocker tetrodotoxin 1 μM and (b) the effect of antagonists for proposed excitatory and inhibitory neurotransmitters on spontaneous rhythmic phasic contractions (RPCs). Y-axis depicts the magnitude of contractions and results are expressed in percentage of basal control values. ^*P<0.05, ^**P<0.01.

Figure 2

The effect of (a) the proposed excitatory neurotransmitters—ACh and substance P (SP) and (b) inhibitory neurotransmitters—the nitric oxide donor sodium nitroprusside (SNP) and the specific P2Y₁ agonist 2MeSATP. Y-axis depicts the magnitude of contractions. ^*P<0.05 vs basal values.

Figure 3

Patterns of electric field stimulation (EFS)-induced responses in strips of human sigmoid colon. Left panel shows an off-contraction appearing briefly following EFS. Right panel shows an EFS-induced on-contraction during EFS. ^*P<0.05 vs 1 Hz; ^#P<0.05 vs off-contractions.

Figure 4

Pharmacological characterization of off-contractions induced by electric field stimulation (EFS). Latency was significantly reduced by N^G-nitro-L-arginine (L-NNA), unaffected by the sequential addition of atropine, and further reduced after the addition of apamin leading to an on-contraction during the electrical stimulus. Amplitude of EFS-induced off-contractions was significantly enhanced by L-NNA, reduced by atropine and the residual contraction was enhanced by sequential addition of apamin ^*P<0.05 statistical significance between the addition of the current and previous drug.

Figure 5

Characterization of non-nitrergic responses in strips showing electric field stimulation (EFS)-induced off-contractions. Strips were incubated with N^G-nitro-L-arginine (L-NNA; 1 mM) and atropine (1 μM) before drug addition. Left panels show quantitative effects and right panels show typical recordings. (a) MRS 2179 (10 μM) significantly reduced both latency and amplitude. (b) NF279 (10 μM) had no effect on EFS-induced responses. (c) α-chymotrypsin (α-CMT; 10 U mL⁻¹) increased the amplitude of off-contractions. ^***P<0.001.

Figure 6

Characterization of electric field stimulation (EFS)-induced (a) off- and (b) on-contractions. Atropine (1 μM) and the NK₂-RA (1 μM) significantly reduced the amplitude of EFS off- and on-contractions. ^*P<0.05, ^***P<0.001.

Figure 7

Presynaptic modulation of electric field stimulation (EFS)-induced contractions. MRS 2179 (10 μM) significantly reduced the amplitude of EFS-induced on-contractions (a). In contrast, MRS 2179 did not alter the direct contraction induced by ACh (b) and substance P (c) on smooth muscle cells. ^*P<0.05.

Figure 8

Nicotine (a) and α-β-methylene-ATP (α-β-met-ATP; b) abolished the spontaneous rhythmic phasic contractions (RPCs) in a concentration-dependent manner. ^*P<0.05 vs basal values.

Figure 9

Inhibition of spontaneous motility induced by nicotine (100 μM) was significantly antagonized by N^G-nitro-L-arginine (L-NNA; 1 mM) and fully blocked by the simultaneous addition of L-NNA and (a) apamin (1 μM) or (b) L-NNA and MRS 2179 (10 μM). ^*P<0.05; ^***P<0.001.

Figure 10

Mechanisms of inhibition of the spontaneous motility induced by α-β-methylene-ATP (α-β-met-ATP; 10 μM). ^*P<0.05; ^***P<0.001.