Role of intracellular calcium in contraction of internal anal sphincter

Abstract

Key Words: intracellular calcium, internal anal sphincter, ryanodine, muscle contraction

INTRODUCTION

Internal anal sphincter (IAS) is a continuation of the smooth circular muscle layer thickened at the rectum, innervated by vegetative nerve. IAS is a special smooth muscle, which is different from colonic smooth muscle in physiology and pharmaology[1]. It was found that contraction of gastric smooth muscle depends on the influx of extracellular calcium and release of intracellular calcium[2]. In present study, we observed and compared the effects of extra and intracellular calcium on the contraction of IAS and colonic smooth muscle.

MATERIALS AND METHODS

IAS and colonic smooth muscle (circular muscle) were taken from 10 patients undergoing abdomino-perineal resection of the rectal carcinoma. The strips containing muscle were approximately 1.2 cm long and 0.2 cm wide. The samples were immediately immersed in Krebs-Henseleit solution. The muscle segments were held by the extremities in the direction of the fires and suspended in a 35mL organ bath containing Krebs-Henseleit solution at 36.5 °C ± 0.1 °C an d gassed with 95% oxygen-5% carbon dioxide. Segments were attached to an isometric transducer coupled to a polygraph recorder.

Responses of the strips to acetylcholine (Ach) and noradrenaline (NA) were examined using cumulative concentration responses. After this, repeated steps were made in calcium-free and calcium solution respectively to test effects of extracellular cacilum on the contraction of the muscle. Finally, ryanodine of different doses was given to the strips in solution containing calcium to study the role of intracellular calcium in contraction of the muscle.

Anal manometry was made in 32 healthy volunteers to obtain the contracting figures of anal sphincter. The average age was 47 years (ranging from 33-61). The manometry consisted of a perfused open - ended polyethylere catheter (inside diameter 1mm) attached to an external transducer (CYS, China), and a micro computer (Laser-310).

Statistical results were compared using Student’s test. P < 0.05 was considered to be significant.

RESULTS

Contracting frequency of colonic smooth muscle and IAS

In vitro spontaneous contraction of 7-12 and 11-17 cycles per min (cpm) could be seen in colonic smooth muscle and IAS respectively. The contracting frequency of anal sphincter from the manometric figures was 12 cpm-18 cpm under the resting condition in these volunteers.

Effects of Ach and NA on colonic smooth muscle and IAS

Ach had a dose-dependent relation with contraction of colonic smooth muscles and had no effect on internal anal sphincter. NA had a dose-dependent relation with contraction of internal anal sphincter and had no effect on colonic smooth muscles (Table 1).

Table 1 Dose-response correlation of Ach and NA in colonic smooth muscle and IAS (^-x±s, n = 10).

Colonic smooth muscle		Internal anal sphincter (IAS)
Ach (mol/L)	Contraction strength (g)	NA (mol/L)	Contraction strength (g)
10-6	1.2 ± 0.39	10-6	0.70 ± 0.23
10-5	2.4 ± 0.41	5 × 10-6	1.34 ± 0.41
10-4	4.1 ± 1.20	10-5	1.74 ± 0.41
10-3	6.4 ± 1.60	5 × 10-5	2.20 ± 0.44
10-2	9.3 ± 1.75	10-4	2.68 ± 0.45

Contracting effects of extracellular calcium on colonic smooth muscle and IAS

Significant difference was found in contractions of colonic smooth muscles in calcium and calcium-free solutions, but no difference was found in contraction of IAS in calcium and calcium-free solutions (Table 2).

Table 2 Contraction of colonic smooth muscle and IAS in different solutions (^-x±s, n = 10).

Solution	Contraction strength (g)
	Colonic smooth muscle	Internal anal sphincter
Ca2+ solution	8.30 ± 2.01	2.44 ± 1.20
Ca2+ -free solution	4.27 ± 0.91a	1.84 ± 0.77

^aP < 0.01, t = 3.72, compared with Ca²⁺ containing solution.

Effects of ryanodine on contraction of colonic smooth muscle and IAS

Ryanodine had a remarkably inhibiting effect on contractions of IAS, but had no effect on contractions of colonic smooth muscles (Table 3).

Table 3 Inhibiting effect of ryandine on colonic smooth muscle and IAS (^-x±s, n = 10).

Ryanodine (μmol/L)	Percentage of contractile strength without ryanodine (%)
	Colonic smooth muscle	Internal anal sphincter (IAS)
0.1a	85 ± 17.9	91 ± 11
1.0	87 ± 13	71 ± 24b
10.0	85 ± 14	57 ± 20c

^bP < 0.05, t = 2.48;

^cP < 0.01, t = 3.61; compared with a.

DISCUSSION

Smooth muscle of gut is innervated by cholinergic and adrenergic nerves. Acetylcholine is effective in contracting colonic smooth muscles, whereas epinephrine inhibits it. It is these nerves that regulate and control the movement of the smooth muscles of digestive tract.

The contracting frequency of IAS is basicaly in accordance with anal sphincter in the volunteers. The contracting frequency of IAS is higher than that of colonic circular muscle. It is possibly related to the maintenance of continence because anal resting pressure is mainly produced by contraction of IAS[3].

It is well known that contraction of muscular cell is associated with Ca²⁺, and contraction of colonic smooth muscle is chiefly produced by influx of Ca²⁺. From present study we found that contraction of colonic smooth muscle was weakened by about 50% when no extracellular calcium exists, whereas contraction of IAS was not affected. It is suggested that releases of Ca²⁺ from intracellular storage may occur at the time when IAS contracts.

Ryanodine is an alkaloid extracted from rhizome of Ryania speciosa Vahl. It has been found in animal experiments that when Ryanodine is combined with Ryanodin receptor of sarcoplasmic reticulum in smooth muscular cells, contraction of the m uscular cells will be prevented because Ca²⁺ storage is exhausted after re lease of Ca²⁺ from it[4-7]. In the present study we found that contraction of IAS induced by agonist was significantly inhibited in contrast with colonic smooth muscle when Ryanodin was given. It is indicated that release of intracellular cacium may play an important role in contraction of IAS. IAS is a special smooth muscle, being principally responsible for the generation and maintence of resting anal canal pressure[3]. It is still unknown whether the contracting property of IAS is associated with the release of intracellular calcium. Futher studies should be made.