Colitis affects the smooth muscle and neural response to motilin in the rabbit antrum

Abstract

Background and purpose: The underlying mechanisms of gastric dysfunction during or after an episode of intestinal inflammation are poorly understood. This study investigated the effects of colitis on the contractile effects of motilin, an important endocrine regulator of gastric motility, in the antrum.

Experimental approach: Myeloperoxidase (MPO) activity, NF-kappaB activity and motilin receptor density were determined in the antrum of rabbits 5 days after the induction of 2,4,6-trinitrobenzenesulphonic acid colitis. Smooth muscle and neural responses to motilin were studied in antral smooth muscle strips in vitro.

Key results: Colitis did not affect MPO activity, but increased NF-kappaB activity in the antrum. Motilin receptor density in the antrum was not affected. Under control conditions, motilin induced a slowly developing tonic smooth muscle contraction. Five days post-inflammation, tonic contractions to motilin were reduced and preceded by a rapid initial contraction. Other kinases were recruited for the phosphorylation of myosin light chain (MLC) (a multi-functional MLC kinase), and for the inhibition of MLC phosphatase (Rho kinase in addition to protein kinase C) to mediate the motilin-induced contractions during inflammation. Colitis potentiated the cholinergic neural on-contractions in the antrum. This was associated with a hyper-reactivity to motilin and an increased muscle response to ACh.

Conclusions and implications: Colitis altered the course of the motilin-induced smooth muscle contraction in the antrum. This involved changes in the kinases phosphorylating MLC. Increased cholinergic excitability to motilin in the antrum may play a role in the pathogenesis of inflammation-associated gastric motility disorders.

Figure 1

Effect of colitis on NF-κB activity in the antrum. (A) Nuclear extracts were prepared from the antrum of control rabbits (lane 1) or from rabbits 5 days after the induction of colitis with TNBS (lane 2), and EMSA was performed. (B) Densitometric analysis using Scion image software (Scion Corp., Frederick, MD, USA) of NF-κB activity in nuclear extracts prepared from the antrum of control (n= 5) and inflamed (n= 8) rabbits. (C) Competition analysis was performed by incubating nuclear extracts from tissue of rabbits with colitis with ³²P NF-κB and a 100-fold excess of unlabelled NF-κB oligonucleotide probe (lanes 3–4), or SP₁ oligonucleotide with a binding site for an unrelated protein (lanes 5–6). The results were compared to experiments to which only ³²P-NF-κB was added (lanes 1–2).

Figure 2

Effect of TNBS colitis on the smooth muscle response to motilin in the antrum. Strips from the antrum of control rabbits or from rabbits 5 days after the induction of colitis were stimulated with 10⁻⁷ M motilin in the presence of 3 µM TTX, and the contractile response was measured isometrically. Control strips responded to motilin with a slowly developing tonic contraction, while in strips from rabbits with colitis the tonic contraction was preceded by a rapid initial contractile response.

Figure 3

Effect of Ca²⁺-dependent and Ca²⁺-independent kinase inhibitors on the sustained smooth muscle contraction to motilin in the antrum from control rabbits (A) and from rabbits 5 days after the induction of TNBS colitis (B). Strips were pre-incubated in the presence of TTX (3 µM) with MLCK inhibitors (10 µM ML-7 or KN-62) involved in the phosphorylation of MLC, or with kinase inhibitors involved in the inhibition of MLC phosphatase (10 µM GF-109203X or HA-1077) before stimulation with motilin (10⁻⁷ M). The results are expressed as a percentage of the response to motilin in the presence of 3 µM TTX, but in the absence of inhibitors. The results are the mean ± SEM (control: n= 7, N= 4; colitis: n= 5, N= 3). *P < 0.05, **P < 0.01, significantly different from responses to motilin in the absence of inhibitors.

Figure 4

Effect of kinase inhibitors involved in the phosphorylation of myosin light chain on the rapid initial smooth muscle contraction to motilin in the antrum from rabbits after the induction of TNBS colitis. Strips were pre-incubated in the presence of TTX (3 µM) with MLCK inhibitors (10 µM ML-7 or KN-62) involved in the phosphorylation of MLC, or with inhibitors of MLC phosphatase (10 µM GF-109203X or HA-1077) before stimulation with motilin (10⁻⁷ M). The results are compared to the response to motilin in the presence of TTX, but in the absence of inhibitors. The results are the mean ± SEM (n= 5, N= 3). *P < 0.01, significantly different from responses to motilin in the absence of inhibitors.

Figure 7

Effect of TNBS-induced colitis on the neural contractile response to motilin in the antrum. Strips from the antrum of control rabbits or from rabbits 5 days after the induction of colitis were stimulated by EFS in the absence or presence of 1 nM motilin, and the mean response during (on-contraction) (A) and after (off-contraction) (B) the stimulation period at different frequencies (1–16 Hz) was determined and expressed in g·mm⁻². The results are the mean ± SEM (control: n= 10, N= 10; colitis: n= 12, N= 10). +P < 0.01, ++P < 0.001, significantly different from responses in strips from control rabbits in the absence of motilin. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from responses in strips from rabbits with colitis in the absence of motilin.

Figure 6

Effect of TNBS-induced colitis on the neural contractions in the antrum. Strips from the antrum of control rabbits or from rabbits 5 days after the induction of colitis were stimulated by EFS, and the mean response during (on-contraction) and after (off-contraction) the stimulation period at different frequencies (1–16 Hz) was determined and expressed in g·mm⁻². The results are the mean ± SEM (control: n= 48, N= 24; colitis: n= 42; N= 21). *P < 0.001, significantly different from on-contraction in strips from control rabbits.

Figure 5

Representative tracings showing the different responses to EFS in antral strips from a control and a TNBS colitis rabbit (A), and the effect of motilin on each response (B). (A) Antral strips from a control rabbit (left) and a rabbit 5 days after the induction of TNBS colitis were stimulated by EFS at increasing frequencies (1–16 Hz). (B) Effect of motilin (1 nM) on neural responses elicited by EFS at 8 Hz of antral strips from a control rabbit (left) and a rabbit with TNBS colitis (right).

Figure 8

Effect of TNBS-induced colitis on the smooth muscle response to ACh, SP or KCl in the antrum. Strips from the antrum of control rabbits or from rabbits 5 days after the induction of colitis were stimulated with 100 µM ACh, 1 µM SP or 60 mM KCl, and the contractile response (g·mm⁻²) was measured isometrically. The results are the mean ± SEM (control: n= 16, N= 7; colitis: n= 16, N= 7). *P < 0.05, **P < 0.001, significantly different from smooth muscle contractions in strips from control rabbits.