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Proceedings of the Society for Experimental Biology and Medicine 224:147-151 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Differential Effects of Maturation on Nicotinic- and Muscarinic Receptor–Induced Ion Secretion in Guinea Pig Distal Colon

Adrianne R. Powell and Rhoda A. Reddix1,


Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The incidence of constipation increases with age. This has been linked to age-related changes in the structure and function of myenteric neurons regulating intestinal motility; however, the role of submucous neurons is unknown. The aim of this study was to determine the effect of maturation on cholinergic receptor–induced ion secretion in guinea pig colon. Changes in the short-circuit current (Isc) and tissue conductance were monitored in muscle-stripped colonic segments from young (3–4-month-old) and mature (12–15-month-old) male guinea pigs. Thirty-one percent of colonic segments from young guinea pigs exhibited ongoing neural activity, which was absent in mature animals. Baseline Isc was significantly higher only in young guinea pig tissues with ongoing activity. Tissue conductance was similar in all tissues. Electrical field stimulation caused a biphasic increase in the Isc. At 15 V/10 Hz, only Peak 1 was attenuated, whereas both peaks were reduced in mature guinea pigs at 10 V/5Hz. 1,1,dimethyl-4-phenyl-piperazinium(DMPP)–induced ion secretion was blunted in mature guinea pigs. Atropine reduced the 1,1,dimethyl-4-phenyl-piperazinium response only in young guinea pigs. Carbachol-induced ion secretion was similar in tissues from both age groups. In conclusion, nicotinic receptor–induced secretion mediated by both cholinergic and noncholinergic secretomotor neurons was blunted; however, epithelial muscarinic receptor activity was unaltered during maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Constipation is commonly observed in the elderly (1). Numerous studies have shown that constipation results primarily from changes in gastrointestinal motility such as chronic intestinal dilation, elongation, irregular smooth muscle contraction, and slowed intestinal transit (2). These changes in gut motility are usually secondary to alterations in extrinsic and/or intrinsic neural control of intestinal smooth muscle.

Several studies demonstrating age-related structural and functional changes in myenteric ganglia regulating gastrointestinal motility have been reported (3-16). Structural changes in the myenteric ganglia of colonic segments isolated from aged rats were first identified by Yamajata (16). This was confirmed by Gabella (7) who showed a reduction in the size of myenteric ganglia and neuron density per myenteric ganglion in the small intestine of aged guinea pigs (26–30 mos). A similar age-associated decline in the number of myenteric neurons of both the small and large intestine has also been reported in rats (14) and in human esophagus and small intestine (9, 11). In addition to decreased ganglion size and neuron count, a loss of substance P, vasoactive intestinal polypeptide, and somatostatin-containing nerve fibers has been observed in the small intestine of aged rats (6).

Results from functional studies provide additional evidence supporting a decline in myenteric neural control of gastrointestinal motility. This is evidenced by the fact that neurally evoked contraction of isolated intestinal smooth muscle preparations was blunted in aged rats (10, 13). Acetylcholine, a major neurotransmitter within myenteric neurons, stimulates intestinal smooth muscle contraction. The authors suggested that the blunted smooth muscle contraction in aged rats was due to a loss of cholinergic neural activity with age (13). This is supported by the fact that acetylcholine release was attenuated in colonic segments isolated from senescent rats compared with young animals. Moreover, the authors showed that the decline in acetylcholine release was due to diminished influx of calcium into nerve terminals upon stimulation (13). Similarly, Burleigh (1) also showed that acetylcholine levels are severely reduced in segments of human colon in subjects experiencing constipation.

In addition to the changes in intestinal motility, age-related changes in enteric neural pathways regulating fluid and electrolyte transport across gut epithelia may represent another contributing factor in the pathophysiology of constipation in the elderly. The submucous plexus is the primary branch of the enteric nervous system that regulates gastrointestinal blood flow and electrolyte transport. However, very little is known concerning the effect of age on submucous neural pathways regulating epithelial ion transport. Since the myenteric and submucous plexuses work in concert to coordinate gastrointestinal function, it seems possible that there may be simultaneous age-associated changes in the structure and function of neurons within both plexuses. The focus of this study was to determine whether there is a similar age-related decline in submucous neural pathways controlling epithelial ion transport to that observed in myenteric neural pathways regulating intestinal motility. Acetylcholine is a major neurotransmitter in submucous secretomotor neurons (17). We hypothesize that a decline in cholinergic receptor-induced ion secretion contributes to the development of constipation. More specifically, we examined changes in nicotinic and muscarinic receptor–evoked ion secretion in submucous/mucosal preparations of colon isolated from young (3–4-month-old) and mature (12–15-month-old) guinea pigs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Preparation.
Young (3–4-month-old) and mature (12–15-month-old) male albino guinea pigs (Harlan Sprague-Dawley, Indianapolis, IN) weighing 350–500 g and 900–1000 g, respectively, were housed in metal cages with food and water ad libitum. The animals were stunned and exsanguinated. This method of euthanasia has been approved by the LSU Institutional Animal Care Committee and complies with federal regulations. Segments of distal colon were removed and opened along the mesenteric border. The intraluminal contents was removed, and the segments were pinned with the mucosal side down onto a Sylgard-coated Petri dish. Tissues were perfused with a chilled Krebs-Ringer solution (in mM): 120 NaCl, 6 KCl, 1.2 MgCl2, 6 H2O, 1.3 NaH2PO4H2, 14.4 NaHCO3, 2.5 CaCl2, and 12.5 glucose. The longitudinal and circular muscle layers along with the myenteric plexus were removed by blunt dissection leaving the submucosa/mucosa intact.

Short-Circuit Current (Isc) Measurements.
Colonic segments were divided into 2–3-cm sheets and mounted in Ussing flux chambers with an area of 0.785 cm2. Tissues were bathed in a Krebs-Ringer solution maintained at 37°C and aerated with 95% O2 and 5% CO2. The chambers were designed with ports for Ringer-agar bridges and calomel half-cells for measurement of transmural potential difference (PD; mV). Throughout the experiment, tissues were short-circuited with a voltage clamp apparatus (Physiological Instruments, San Diego, CA) to abolish changes in PD. Any changes in the short-circuit current (µA/cm2) during either electrical or chemical stimulation of tissues are due to alterations in active ion transport. Tissue conductance (Gt; mS/cm2) was calculated according to Ohm's law. Tissues were equilibrated for 40 min, and baseline Isc, Gt, and PD were recorded

Electrical Field Stimulation (EFS).
Each tissue was electrically stimulated for 90 sec by a pair of aluminum foil electrodes placed adjacent to the serosal surface. The electrodes were connected to a Grass SD 88 stimulator (Grass Instruments, Quincy, MA) that generated pulses at 0.5 ms, with a strength of 10 or 15 V and frequencies of either 5 or 10 Hz. Changes in Isc were continuously monitored by a Kipp and Zonen (Delft, Holland) chart recorder. Measurements of Isc were calculated as the difference between the peak changes in Isc and baseline Isc before stimulation.

Effect of Carbachol and 1,1,dimethyl-4-phenyl-piperazinium (DMPP) on Baseline Short-Circuit Current (Isc) Measurements.
Changes in Isc were monitored in colonic segments exposed to vehicle, 0.1 mM carbachol, a muscarinic receptor agonist or the nicotinic receptor agonist, 0.1 mM DMPP. To determine whether DMPP-induced activation of both cholinergic and noncholinergic neurons were affected to the same degree, another set of tissues was pretreated with the muscarinic receptor antagonist, atropine (10 µM) and then challenged with DMPP. At the end of each experiment, carbachol was added to confirm blockade of muscarinic receptors. The doses of each drug have previously been shown to elicit maximal changes in Isc (18-19).

Chemicals.
Atropine, carbachol, and DMPP were purchased from Sigma Chemical Co. (St Louis, MO). All drugs were dissolved in Krebs-Ringer solution.

Statistics.
All data were expressed as means ± SEM. An unpaired Student's t test was used to test the significance between group means. A probability value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table IGo shows the baseline changes in short-circuit current (Isc), tissue conductance (Gt), and potential difference (PD) in colonic segments isolated from young and mature guinea pigs. Thirty-one percent of muscle-stripped colonic segments from young guinea pigs exhibited ongoing neural activity. Ongoing neural activity is characterized as tetrodotoxin-sensitive oscillations in baseline short-circuit current (20). The baseline Isc was higher in tissues with ongoing activity from young animals than tissues without ongoing activity from both age groups.


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  		Table I.   Baseline Short-Circuit Current in Guinea Pig Colon
 
Electrical field stimulation (EFS) evoked a biphasic increase in Isc in tissues isolated from both groups. It was previously shown that in guinea pig colon, Peaks 1 and 2 of the EFS response represent cholinergic and peptidergic/cholinergic–induced chloride secretion, respectively (21). As shown in Figure 1AGo, at EFS = 15 V and 10 Hz, the initial cholinergic peak was attenuated in colonic segments from mature guinea pigs as compared with the young group. However, Peak 2 of the EFS response was unaltered with age. However, both peaks were attenuated in colonic preparations from mature animals at EFS = 10 V/5 Hz (Fig. 1B)Go.



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  		Figure 1.   Electrical field stimulation (EFS)–induced changes in baseline short-circuit current (Isc; µA/cm2) in muscle-stripped colonic segments isolated from young (3–4-month-old) and mature (12–15-month-old) guinea pigs. (A) EFS = 15 V/10 Hz and (B) EFS = 10 V/5 Hz. Values are reported as the mean ± SEM; n = 10–11; *, significantly different from pK1 in young guinea pig; **, significanly different from pK2 in young guinea pig colon at P < 0.05.

 
To examine the possibility that the reduction in cholinergically mediated secretion may be due to blunted nicotinic receptor responses, colonic segments from each age group were exposed to the nicotinic receptor agonist, DMPP, (0.1 mM). The DMPP-induced secretory response was attenuated in tissues from mature animals compared with young guinea pigs. Since both peaks of the EFS response were reduced at lower stimulus strength and frequency, we investigated the possibility that DMPP activation of cholinergic and noncholinergic neurons was altered to the same extent with age. The tissues were pretreated with atropine to allow examination of age-related changes in noncholinergic neural pathways. Atropine decreased the DMPP response by 72% in tissues isolated from young animals but had no significant effect on mature guinea pigs (Fig. 2)Go.



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  		Figure 2.   Changes in the short-circuit current (Isc; µA/cm2) in muscle-stripped colonic segments isolated from young (3–4-month-old) and mature (12–15-month-old) guinea pigs. Tissues were exposed to the nicotinic receptor agonist, DMPP (0.1 mM) in the absence and presence of the muscarinic receptor antagonist, atropine (ATR; 10 µM). Values are expressed as the mean ± SEM; n = 4–7. *, significantly different from young guinea pig colon in the absence of atropine; **, significantly different from mature guinea pig colon in the absence of atropine at P < 0.05.

 
To determine whether the reduced cholinergic response was due to a decline in muscarinic receptor responsiveness, all tissues were challenged with the muscarinic receptor agonist, carbachol (Carb, 0.1 mM). Carbachol caused a similar increase in the Isc in colonic preparations isolated from both age groups (Fig. 3)Go. The carbachol response was abolished by pretreatment with atropine (ATR; 10 µM).



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  		Figure 3.   Changes in the short-circuit current (Isc; µA/cm2) in muscle-stripped colonic segments isolated from young (3–4-month-old) and mature (12–15-month-old) guinea pigs exposed to the muscarinic receptor agonist, carbachol (Carb; 0.1 mM). Young: 361.6 ± 52.2 µA/cm2 and mature: 392.6 ± 53 µA/cm2. All values are expressed as the mean ± SEM; n = 10–11.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Maturation is defined as the period during which physiological processes are fully developed. It immediately precedes the aging process. Hence, the onset of age-related changes in gastrointestinal function may begin during maturation and continue as the individual ages. Previous studies have clearly demonstrated age-related structural and functional changes in myenteric neural pathways of various species. Results from the current study suggest that there is also a corresponding decline in submucous neural control of colonic ion secretion in aged guinea pigs. More specifically, our results showed that there is a decline in nicotinic receptor–induced activation of secretomotor neurons with age. Taken together, these results suggest that a decline in secretomotor activity would promote ion absorption thereby contributing to the development of constipation in the elderly.

In a previous study, we reported that in vitro preparations of guinea pig colon exhibit basal ongoing neural activity (22). We also observed basal neural activity in 31% of colonic segments from young guinea pigs. However, this neural activity was absent in mature animals. The baseline short-circuit current was significantly higher in young guinea pigs with ongoing neural activity than in tissues without ongoing activity in both age groups. It has previously been reported that electrical field stimulation of muscle-stripped guinea pig colon causes a biphasic increase in Isc characterized by an initial cholinergic Peak 1 followed by a second peak mediated primarily by mixed cholinergic and peptidergic neurotransmitter release (20). We observed a similar pattern in our preparations. The results showed a significant reduction in Peak 1 (cholinergic component) in mature animals compared with the young group. However, Peak 2 was unaltered. Both peaks were attenuated in colonic segments from mature animals, at 10 V/5 Hz (lower stimulus and frequency). These results taken together suggest that both cholinergic and noncholinergic secretomotor activity is altered during maturation. However, the cholinergic neural activity is altered to a greater extent than noncholinergic neurons.

Acetylcholine evokes ion secretion in guinea pig colon via activation of both muscarinic receptors localized primarily on colonocytes (17, 20, 22). Muscarinic receptors have also been identified on submucous neurons of guinea pig intestine (22). Additionally, acetylcholine may activate nicotinic receptors on cell bodies of interneurons and secretomotor neurons with a subsequent release of cholinergic and peptidergic neurotransmitters from these neurons. To determine whether maturation alters nicotinic receptor-induced secretion, we examined the effect of the nicotinic agonist, DMPP on baseline Isc in tissue from both age groups. There was an 85% reduction in the DMPP response in mature guinea pigs compared with young animals. This suggests a reduction in nicotinic responsiveness possibly characterized by a reduction in the number of membrane-bound receptors, a reduction in the number of functional receptors without changes in receptor number, or a decrease in neurotransmitter release upon stimulation. Non-cholinergic neurons represent 50% of the submucous secretomotor neuronal population. Peak 2, which is composed primarily of noncholinergic neurons, was reduced only at a lower stimulus strength and frequency. This result indicated that there is a small alteration in the activation of noncholinergic secretomotor neurons.

Since the greatest age-related reduction was observed in Peak 1 (cholinergic), we examined the effect of atropine (muscarinic receptor antagonist) on DMPP-induced secretion to determine whether the cholinergic-mediated secretory response was reduced in the mature animal. The results showed that atropine reduced the DMPP response by 72% in young guinea pigs but had no effect on mature guinea pigs. This result provides strong evidence that cholinergically mediated secretion is blunted whereas there are small changes in noncholinergic neural activity. The blunted cholinergic response was not due to altered epithelial muscarinic receptor activation because the muscarinic receptor agonist, carbachol, stimulated chloride secretion to a similar degree in both age groups. Based on these data, we hypothesize that the blunted cholinergic response was due to a decline in acetylcholine release from secretomotor neurons. This proposed idea is currently under investigation in our laboratory.

In conclusion, our results suggest that the role of myenteric neural pathways regulating motility has received considerable attention as a key factor in the development of constipation during aging (3-16). However, there are no studies to date demonstrating age-related changes in submucous secretomotor neurons and intestinal function. Our results suggest that both cholinergic and noncholinergic secretomotor neural activities decline during maturation. However, the greatest decline was observed in cholinergic secretomotor neural activity. More specifically, nicotinic receptor–induced secretion was reduced without changes in muscarinic receptor–induced secretion. The results indicate that the decline in secretomotor function promotes ion absorption and therefore may contribute to the development of constipation in the aging gut.


   Acknowledgments
 
The authors would like to thank Mrs. Xiao-Mei Niu for her technical assistance with the short-circuit current measurements.


   Footnotes
 
Funding was received through the Louisiana Education Quality Support Fund RD-A-17.

1 To whom requests for reprints should be addressed at the Department of Pharmacology & Experimental Therapeutics, LSU Health Sciences Center, New Orleans, LA 70112. E-mail: rreddi{at}lsumc.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burleigh DE. Evidence for a functional cholinergic deficit in human colonic tissue resected for constipation. J Pharm Pharmacol 40:55–57, 1988.[Medline]
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  14. Santer RM, Baker DM. Enteric neuron numbers and sizes in Auerbach's plexus in the small and large intestine of adult and aged rats. J Auton Nerv Syst 25:59–67, 1988.[Medline]
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Received for publication June 16, 1999. Accepted for publication February 10, 2000.




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