The mechanisms underlying the generation of the colonic migrating motor complex in both wild-type and nNOS knockout mice

Abstract

Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS)(-/-) mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS(-/-) mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 +/- 0.6 mV; nNOS(-/-): 11.2 +/- 1.5 mV). Both atropine (1 microM) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 microM] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 microM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS(-/-) mouse colon (wild-type: 47.9 +/- 2.4 mm; nNOS(-/-): 57.8 +/- 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks.

Fig. 1.

Simultaneous electrical and tension recordings during the colonic migrating motor complex (CMMC) in wild-type colon. A: intracellular microelectrode recordings (electro) were made from circular muscle (CM) cells in the midcolon. Electrical activity, which consisted of fast electrical oscillations and action potentials (Inset) during the CMMC, was tightly coupled to contraction of the CM. B: CMMCs were recorded from longitudinal muscle (LM) cells in the midcolon that occurred at the same time as a CMMC contraction in the CM. Note the fact that the longitudinal muscle is more depolarized than the CM. C: effects of blocking nitric oxide synthesis with N^ω-nitro-l-arginine (l-NA) on electrical and mechanical activity.

Fig. 2.

CMMCs in neuronal nitric oxide synthase (nNOS)^−/− mouse colon. A: simultaneous electrical and mechanical activity of the CM. Note the underdeveloped middle complex. Inset: nNOS is expressed only in the brain and colon of wild-type mice but not in the nNOS^−/− mouse. HPRT, hypoxanthine guanine phosphoribosyl transferase; eNOS, endothelial NOS; iNOS, inducible NOS. B: tension recordings at 2 sites along the colon 4 cm apart. l-NA increased the frequency of contractions in the wild-type colon but had little effect in the nNOS^−/− mouse. To, oral tension transducer; Ta, anal tension transducer. C: spatiotemporal maps show that pellet propulsion was faster in the wild-type colon (left) compared with that in the nNOS^−/− mouse (right).

Fig. 3.

Effect of cholinergic and tachykinin antagonists on the CMMC in wild-type mice. A: control CMMC with associated contraction. B: effect of atropine on the CMMC. Note that the fast oscillations and action potentials were present; however, atropine reduced the contraction. C: atropine plus the addition of MEN 10,376 further reduced the area under the CMMC and its associated contraction although the fast oscillations and action potentials were still present. D: following the further addition of RP 67580, the fast oscillations, action potentials, and contraction were abolished. However, following the addition of the neurokinin (NK)1 antagonist, periodic bursts of inhibitory junction potentials were observed (see arrow). E: further addition of l-NA revealed fast oscillations and fast inhibitory junction potentials in the CM near the myenteric border. F: further addition of apamin revealed robust slow-wave activity. G: summary of the effects of atropine and tachykinin antagonists on the area under the slow depolarization during the CMMC (n = 10); *P < 0.05, **P < 0.01.

Fig. 4.

Effect of cholinergic and tachykinin antagonists on the CMMC in nNOS^−/− mice. A: control CMMC with associated contraction. Note that the muscle was more depolarized than in the wild-type mice. B: atropine reduced the amplitude of the fast oscillations and spiking activity during the CMMC and its associated contraction. C: atropine plus the addition of MEN 10,376 further reduced the area under the CMMC, the fast oscillations, and its associated contraction although the fast oscillations and action potentials were still present. D: following the further addition of apamin, slow waves with action potentials occurred, giving rise to phasic contractions of the muscle.

Fig. 5.

Effect of cholinergic and tachykinin antagonists on the evoked CMMC in colons without the mucosa. A: transmural nerve stimulation (0.5-ms pulses at 20 Hz for 1 s, 50 V) evoked robust CMMCs in both wild-type (left) and nNOS^−/− (right) mice. B–D: atropin (B), atropine plus NK2 (C), and atropine plus NK2 and NK1 (D) receptor antagonists had similar effects to these drugs on spontaneous CMMCs. E: summary of the effects of atropine and tachykinin antagonists on the area under the slow depolarization during the CMMC in wild-type mice (n = 10); *P < 0.05, **P < 0.01.

Fig. 6.

Types of slow-wave activity observed following blockade of inhibitory nerves. Following the addition of l-NA (10 μM) and apamin (0.1 μM), 3 types of electrical oscillations were observed in the CM (atropine and the tachykinin antagonists were present). A and B: slow waves, which had a frequency of ∼16 cycles/min, were recorded in CM cells near the submucosal border. When they generated action potentials, they gave rise to phasic contractions. C: A less frequent slow-wave activity (∼6 cycles/min) was also observed in deeper CM cells. D: sometimes both these activities were superimposed on one another. E: in CM near the myenteric border, we observed fast electrical oscillations (∼35 cycles/min). F: slow-wave activity recorded from the longitudinal muscle occurs at the same frequency as contractions in the CM. G and H: following nicardipine, slow-wave activity was blocked.

Fig. 7.

Excitatory control of smooth muscle and interstitial cells of Cajal (ICC) during the CMMC. Ascending interneurons activate excitatory motor neurons that release ACh and tachykinins (TK) to stimulate myenteric ICC and submucosal ICC. ACh and tachykinins activate muscarinic (M) receptors and NK2 receptors on muscle cells and muscarinic and NK1 receptors on ICC (adapted from Ref. 11).