Rhythmic changes in colonic motility are regulated by period genes

Abstract

Human bowel movements usually occur during the day and seldom during the night, suggesting a role for a biological clock in the regulation of colonic motility. Research has unveiled molecular and physiological mechanisms for biological clock function in the brain; less is known about peripheral rhythmicity. This study aimed to determine whether clock genes such as period 1 (per1) and period2 (per2) modulate rhythmic changes in colonic motility. Organ bath studies, intracolonic pressure measurements, and stool studies were used to examine measures of colonic motility in wild-type and per1per2 double-knockout mice. To further examine the mechanism underlying rhythmic changes in circular muscle contractility, additional studies were completed in neuronal nitric oxide synthase (nNOS) knockout mice. Intracolonic pressure changes and stool output in vivo, and colonic circular muscle contractility ex vivo, are rhythmic with greatest activity at the start of night in nocturnal wild-type mice. In contrast, rhythmicity in these measures was absent in per1per2 double-knockout mice. Rhythmicity was also abolished in colonic circular muscle contractility of wild-type mice in the presence of N(omega)-nitro-L-arginine methyl ester and in nNOS knockout mice. These findings suggest that rhythms in colonic motility are regulated by both clock genes and a nNOS-mediated inhibitory process and suggest a connection between these two mechanisms.

Fig. 1.

Measures of colonic motility are rhythmic in wild-type (WT) mice on a light-dark cycle. A: stool output in WT mice (n = 3). ZT, Zeitgeber time, whereby ZT0 refers to the time at which lights came on and ZT12 refers to the time that lights went off. B: total stool output during light (ZT0–12) and dark cycle (ZT12–0). C: colonic circular muscle contractility in WT mice at ZT1 (n = 4), ZT7 (n = 6), ZT13 (n = 8), and ZT19 (n = 5) expressed as area under the curve (AUC; g·s) ± SE. D: cosinor analysis of colonic circular muscle contractility. PR, percentage rhythm.

Fig. 2.

Rhythmicity in measures of colonic motility in WT mice persists in constant darkness. A: stool output in WT mice (n = 3). B: total stool output during light (ZT0–12) and dark cycle (ZT12–0). C: colonic circular muscle contractility in WT mice at ZT1 (n = 4) and ZT13 (n = 4) expressed as AUC (g·s) ± SE. D: intracolonic pressure changes in a single WT mouse under constant darkness.

Fig. 3.

Rhythmic changes in colonic motility are attenuated in per1per2 knockout mice. A: stool output in per1per2 double-knockout mice (n = 5). B: colonic circular muscle contractility in per1per2 double-knockout mice at ZT1 (n = 4) and ZT13 (n = 4) expressed as AUC (g·s) ± SE. C: intracolonic pressure changes in per1per2 double-knockout mouse under constant darkness.

Fig. 4.

Colonic circular muscle contractility in per1 and per2 knockout mice. Colonic circular muscle contractility in per1 single-knockout mice (A) and per2 single-knockout mice (B) at ZT1 (n = 4) and ZT13 (n = 4) expressed as AUC (g·s) ± SE. NS, not significant.

Fig. 5.

Rhythmic changes in colonic contractile response are neuronally mediated. Colonic circular muscle contractility in WT mice in the absence (−) (n = 4) and presence (+) of TTX (n = 6) (A), in the absence (−) (n = 4) and presence (+) (n = 4) of N^ω-nitro-l-arginine methyl ester (l-NAME; B), in nNOS knockout mice (n = 6) (C), and total stool output during ZT0–12 and ZT12–0 in WT and nNOS knockout mice (n = 3) (D).

Fig. 6.

Proposed paradigm for clock gene-mediated inhibitory effects on colonic motility. Clock genes within the central clock in the brain (a) and within peripheral tissues (c) participate in a complex feedback loop mechanism of cyclical transcription and translation over the course of 24 h [a simplified schematic is presented; detailed review by Bell-Pedersen et al. (3)]. The central clock in the brain synchronizes itself to the light-dark and dictates the phase of peripheral clock genes. The mechanism through which this occurs remains unknown (b). Peripheral clock gene expression lags ∼6–8 h behind the phase of the central clock. Peripheral clock genes will initiate transcription of organ-specific genes through the binding of the CLOCK-Bmal heterodimer complex to a promoter region (direct clock-controlled transcription), which can in turn initiate the transcription of another gene (indirect clock-controlled transcription). It is unknown whether colonic nNOS is under direct or indirect colonic clock control (as indicated by the dotted arrow), but its rhythmic expression mediates an inhibitory effect on colonic motility that coincides with the start of the light cycle.