Age-related changes in melatonin release in the murine distal colon

Abstract

Constipation and fecal impaction are conditions of the bowel whose prevalence increases with age. Limited information is known about how these conditions manifest; however, functional deficits are likely to be due to changes in signaling within the bowel. This study investigated the effects of age on colonic mucosal melatonin (MEL) release and the consequences this had on colonic motility. Electrochemical measurements of MEL overflow demonstrated that both basal and mechanically stimulated MEL release decreased with age. The MEL/serotonin also decreased with increasing age, and the trend was similar to that of MEL overflow, suggestive that age-related changes were primarily due to a reduction in MEL levels. Levels of N-acetylserotonin and the N-acetylserotonin/serotonin ratio were reduced with age, providing an explanation for the reduction in MEL release. Decreases in colonic motility were observed in animals between 3 and 24 months old. Exogenous application of MEL could reverse this deficit in aged colon. In summary, we propose that the age-related decline in MEL release may be due to either decreases or alterations in mechanosensory channels and/or a loss in levels/activity of the N-acetyltransferase enzyme responsible for the synthesis of N-acetylserotonin. Decreases in MEL release may explain the decreases in colonic motility observed in 24 month old animals and could offer a new potential therapeutic treatment for age-related constipation.

Figure 1

Electrochemical determination of melatonin release from distal colon mucosa. Monitoring of all electroactive substances released using differential pulse voltammetry from the mucosa (A) showed the presence of serotonin and melatonin, when compared to standards. Melatonin was monitored amperometrically by obtaining the current difference between +650 and +800 mV vs Ag|AgCl, where DA is dopamine, NE is norepinephrine, and Try is tryamine. (B) Gray bar indicates the duration the BDD electrode was positioned 0.1 mm over the mucosa. Current responses from 3, 12, 18, and 24 month old animals are shown in (C). The population data for the melatonin is shown in (D). The melatonin to serotonin ratio is shown in (E). Data shown as mean ± SEM, n = 6, *p < 0.05 and **p < 0.01 vs 3 month old animals and ^††p < 0.01 and ^†††p < 0.001 vs 12 month old animals.

Figure 2

Alterations in mechanically driven melatonin release with age. The protocol utilized for recordings is shown in (A). Recordings are taken before (zone 1), during (zone 2), and after (zone 3) mechanical stimulation. The white dot indicates the point against which responses are normalized and also indicates the initiation of mechanical stimulation. Current responses from 3, 12, 18, and 24 month old animals are shown in (B). The gray bar indicates the time frame when the glass capillary was utilized to mechanically stimulate villi located adjacent to the BDD electrode. The overall response from multiple animals of all age groups is shown in (C), where the mechanically stimulated melatonin release is shown between 0 and 20 s, and the post mechanically stimulated response shown between 20 and 40 s. The stars indicate the points on the trace utilized for statistical comparison. Data shown as mean ± SEM, n = 5.

Figure 3

Levels of N-acetylserotonin monitored using high performance liquid chromatography. In (A), chromatographic responses of the standards (top trace) and a sample trace from the colonic mucosa of a 3 month old (bottom trace) and 24 month old animal (middle trace) are shown. In (B), the responses from 3 and 24 month old animals are shown at a higher resolution. The amount of N-acetylserotonin found within the mucosa tissue from 3, 12, 18, and 24 month old animals is shown in (C). The ratio of N-acetylserotonin:serotonin is shown for all age groups in (D). Data shown as mean ± SEM, n = 6, *p < 0.05, **p < 0.01, ***p < 0.001 vs 3 month old animals, ^††p < 0.01 and ^†††p < 0.001 vs 12 month old animals, and ^‡‡p < 0.01 vs 18 month old animals. Solutes: (1) 5-hydroxytryptophan, (2) serotonin, (3) tryptophan, (4) 5-hydroxyl indole acetic acid, and (5) N-acetylserotonin.

Figure 4

Alterations in fecal motility with age. Representative traces of fecal pellet motility are shown for 3 and 24 month old animals in (A). The white dot on the pellet indicates the point tracked during recordings, and the linear trace shown indicates the movement of the fecal pellet from the oral to the anal end of the colon. The black bar indicates the duration for which the population data was obtained between multiple animals. The overall data for 3 and 24 month old animals is shown in (B), where the movement of the pellet over 20 min at 2 min intervals is shown. Data shown as mean ± SEM, n = 5, *p < 0.05 and **p < 0.001 3 month old vs 24 month old animals.

Figure 5

Influence of endogenous melatonin on fecal motility with age. Representative traces of fecal pellet motility are shown for 3 and 24 month old animals in (A) and (B), respectively. The white dot on the pellet indicates the point tracked during recordings, and the linear trace shown indicates the movement of the fecal pellet from the oral to the anal end of the colon. The black bar indicates the duration for which the population data was obtained between multiple animals. The overall data for 3 month old animals is shown in (C) and for 24 month old animals in (D), where the movement of the pellet over 14 min at 2 min intervals is shown. Data shown as mean ± SEM, n = 5, *p < 0.05 and **p < 0.001 control vs 1 μM luzindole, ^†p < 0.05, ^††p < 0.01, and ^†††p < 0.001 control vs 1 μM melatonin.

Figure 6

Alterations in melatonin signaling with aging. (A) Proposed signaling mechanism in 3 month animals and how this alters in 24 month old animals (B). Decreases in melatonin release are due to a reduction in the turnover of N-acetylserotonin, and there is a loss in mechanically evoked MEL release. Such changes may explain the age-related reductions in motility observed with age.