Neural mechanisms underlying migrating motor complex formation in mouse isolated colon

Abstract

1. Little is known about the intrinsic enteric reflex pathways associated with migrating motor complex (MMC) formation. Acetylcholine (ACh) mediates the rapid component of the MMC, however a non-cholinergic component also exists. The present study investigated the possible role of endogenous tachykinins (TKs) in the formation of colonic MMCs and the relative roles of excitatory and inhibitory pathways. 2. MMCs were recorded from the circular muscle at four sites (proximal, proximal-mid, mid-distal and distal) along the mouse colon using force transducers. 3. The tachykinin (NK(1) and NK(2)) receptor antagonists SR-140 333 (250 nM) and SR-48 968 (250 nM) reduced the amplitude of MMCs at all recording sites, preferentially abolishing the long duration contraction. Residual MMCs were abolished by the subsequent addition of atropine (1 microM). 4. The neuronal nitric oxide synthase inhibitor, N(omega)nitro-L-arginine (L-NOARG, 100 microM), increased MMC amplitude in the distal region, whilst reducing the amplitude in the proximal region. In preparations where MMCs did not migrate to the distal colon, addition of L-NOARG resulted in the formation of MMCs. Subsequent addition of apamin (250 nM) or suramin (100 microM) further increased MMC amplitude in the distal region, whilst suramin increased MMC amplitude in the mid-distal region. Apamin but not suramin reduced MMC amplitude in the proximal region. Subsequent addition of SR-140 333 and SR-48 968 reduced MMC amplitude at all sites. Residual MMCs were abolished by atropine (1 microM). 5. In conclusion, TKs, ACh, nitric oxide (NO) and ATP are involved in the neural mechanisms underlying the formation of MMCs in the mouse colon. Tachykinins mediate the long duration component of the MMC via NK(1) and NK(2) receptors. Inhibitory pathways may be involved in determining whether MMCs are formed.

Figure 1

Effect of NK₁ and NK₂ receptor antagonists, SR-140 333 and SR-48 968, on MMC amplitude and integral. Addition of the NK₁ and NK₂ receptor antagonists SR-140 333 (250 nM) and SR-48 968 (250 nM) significantly reduced MMC amplitude (a) and integral (b) in all regions of the colon (t-test, P<0.05, n=7). The effects of SR-140 333 and SR-48 968 are expressed as percentages of control MMCs.

Figure 2

Typical recordings demonstrating the effects of NK₁ and NK₂ and muscarinic receptor antagonists SR-140 333 and SR-48 968 and atropine on MMC characteristics. (a) Upper trace: Recording of a control MMC from the proximal-mid region of the colon consisting of rapid contractions with an underlying long duration contraction. Lower trace: Addition of SR-140 333 (250 nM) and SR-48 968 (250 nM) abolished the long duration contraction with the rapid contractions remaining. Recordings were from the same preparation. (b) Upper trace: Recording of a control MMC from the proximal-mid region of the colon consisting of rapid contractions with an underlying long duration contraction. Lower trace: Addition of atropine (1 μM) results in abolition of the rapid contractions with the underlying long duration contraction remaining. Recordings were from the same preparation. Effects of SR-140 333 and SR-48 968 and atropine are shown 15 min after their addition.

Figure 3

Recordings demonstrating the effects of NK₁, NK₂ and muscarinic receptor antagonists, SR-140 333 and SR-48 968 and atropine, added sequentially on MMC characteristics. (a) Recording of a control MMC from the proximal-mid region of the colon consisting of rapid contractions with an underlying long duration contraction. (b) Addition of the NK₁ receptor antagonist SR-140 333 (250 nM) significantly reduced the amplitude and integral of the long duration contraction associated with the MMC. (c) Addition of the NK₂ receptor antagonist SR-48 968 (250 nM), in the presence of SR-140 333, abolished the residual long duration contraction associated with the MMC. (d) Addition of the muscarinic receptor antagonist atropine (1 μM), in the presence of SR-140 333 and SR-48 968 abolished the residual MMC composed of rapid contractions. Recordings were from the same preparation. Effects of SR-140 333, SR-48 968 and atropine are shown 15 min after their addition. Scale bars apply throughout.

Figure 4

Typical recordings displaying the effects of sequential addition of L-NOARG, apamin, SR-140 333 and SR-48 968 and atropine, on MMCs in the proximal, proximal-mid, mid-distal and distal regions of the colon. L-NOARG (100 μM) caused a significant increase in amplitude and integral of MMCs in the distal region, whilst decreasing amplitude and integral in the proximal region. Subsequent addition of apamin (250 nM) caused a further increase in amplitude and integral of MMCs in the distal region, whilst reducing MMC amplitude in the proximal region. Both the rapid and long duration contractions of the MMCs remain after the addition of L-NOARG and apamin. Subsequent addition of SR-140 333 (250 nM) and SR-48 968 (250 nM) significantly reduced MMC amplitude and integral in all regions of the colon whilst preferentially abolishing the long duration component of MMCs. Residual MMCs were abolished by the addition of atropine (1 μM). L-NOARG, apamin and SR-140 333 and SR-48 968 had no effect on MMC frequency in any of the regions. Recordings were made from the same preparation. Scale bars for each region apply throughout. Addition of each drug occurred at least 15 min before the start of the recording shown.

Figure 5

Typical recordings demonstrating the effects of L-NOARG on preparations where MMCs did not propagate to the distal region of the colon. (a) Recording of a preparation lacking MMC activity in the distal region of the colon. (b) Addition of L-NOARG (100 μM; 20 min prior to the start of the recording shown) resulted in MMCs propagating to this region of the colon. Recordings were from the same preparation. Scale bars apply to both (a) and (b).

Figure 6

Typical recordings demonstrating the effects of L-NOARG, suramin, SR-140 333 and SR-48 968 and atropine, added sequentially to the organ bath, on MMCs in the proximal, proximal-mid, mid-distal and distal regions of the colon. L-NOARG (100 μM) caused a significant increase in amplitude and integral of MMCs in the distal region, whilst decreasing amplitude and integral in the proximal region. The subsequent addition of suramin (100 μM) caused a further increase in amplitude and integral of MMCs in the distal region, whilst increasing amplitude and integral in the mid-distal region. Both the rapid and long duration contractions of the MMCs were formed after the addition of L-NOARG and suramin. Subsequent addition of SR-140 333 (250 nM) and SR-48 968 (250 nM) significantly reduced MMC amplitude and integral in all regions of the colon whilst preferentially abolishing the long duration component of MMCs. Residual MMCs were abolished by the addition of atropine (1 μM). L-NOARG, suramin and SR-140 333 and SR-48 968 had no effect on MMC frequency in any of the regions. Recordings were made from the same preparation. Scale bars for each region apply throughout. Addition of each drug occurred at least 15 min before the start of the recording shown.

Figure 7

Typical recording demonstrating the effect of hexamethonium and L-NOARG on MMCs. Addition of hexamethonium (500 μM) abolishes MMC formation in all regions of the colon, as well as causing a small increase in the resting tone of the preparation. The subsequent addition of L-NOARG (100 μM) causes a further increase in the resting tone and the formation of short duration spontaneous contractions.

Figure 8

Typical recording demonstrating the effect of hexamethonium on MMCs in the presence of L-NOARG and atropine. In the presence of L-NOARG (100 μM; added 45 min prior to the start of the recording) and atropine (1 μM; added 15 min prior to the start of the recording), the addition of hexamethonium (500 μM) abolished the residual (long duration) MMCs in all regions of the colon.