Role Of Cholinergic Signal Mediated By Muscarinic Receptors On ICCs In Smooth Muscle Spontaneous Contraction Of Guinea Pig Ureter

Background and Objective:Interstitial cells of Cajal are the special cells of gastrointestinal tract, which pacing the slow waves of gastrointestinal tract, transmit electrical signals, and mediate signals conduction and integration between nerve cells and smooth muscle cells. As an important factor of adjusting the rhythmic contraction of gastrointestinal smooth muscle, interstitial cells of Cajal have close correlation with some gastrointestinal motility disorders, such as constipation, intestinal pseudo-obstruction. Initially, interstitial cells of Cajal were supposed to exist only in gastrointestinal tract. Recently, interstitial cells of Cajal had been reported existed in other organizations outside gastrointestinal tract. Recent researches showed that morphological and immunological features of interstitial cells of Cajal outside gastrointestinal tract resemble those of gastrointestinal interstitial cells of Cajal. So, interstitial cells of Cajal outside gastrointestinal tract that had been called ICC-like cells were collectively referred to as interstitial cells of Cajal (ICCs) now.Under normal circumstances, ureteral smooth muscle similar to gastrointestinal smooth muscle, has spontaneous myogenic contraction uncontrolled by nerve. However, the mechanism of spontaneous myogenic stimulation had not yet been fully illuminated. There was a study demonstrated ICCs been located in the entire urinary tract of human and guinea pig. Solari et al had compared the density of ICCs of renal pelvis and ureter between children with congenital ureteropelvic junction obstruction and normal children, and found that the distribution of ICCs of renal pelvis and ureter of normal children was scattered. The number of ICCs of renal pelvis and ureter of normal children was significantly decreased compared with that of children with congenital ureteropelvic junction obstruction. The study suggests that ICCs have close correlation with origin and regulation of ureter's excitation. Therefore, to further research the role and neuromodulation of ICCs in smooth muscle spontaneous contraction of ureter is helpful to elucidate the role and mechanism of ICCs in the regulation of function of ureteral smooth muscle, and to treat the functional disorders of upper urinary tract.In this study, we compared the smooth muscle spontaneous contraction of isolated guinea pig ureter by muscle strips experiment before and after the effect of carbachol, a nonspecific agonist of muscarinic receptor. Furthermore, we observed if the smooth muscle spontaneous contraction of isolated guinea pig ureter and its reaction to carbachol had been changed after blocking ICCs. Lastly, immunofluorescence (double-labeled) were used to study the expressions of muscarinic receptor subtypes on ICCs of guinea pig ureter. These results will provide new experimental evidences for elucidating the role of cholinergic signal mediated by muscarinic receptors on ICCs in smooth muscle spontaneous contraction of guinea pig ureter.Materials and Methods:The cleaning grade healthy Hartley guinea pigs were sacrificed in this study. Firstly, we investigated the effect of carbachol to smooth muscle spontaneous contraction of guinea pig ureter and the role of ICCs in this effect. 20 guinea pigs were randomly divided into two groups: normal group (n=10) and ICCs inhibited group (n=10). The smooth muscle strips obtained from guinea pigs of ICCs inhibited group were incubated with and measured in Kreb's solution containing 10-4mol/L Glivec, but those of normal group were incubated with and measured in Kreb's solution uncontaining Glivec. All of smooth muscle strips were about 1.0 centimeter obtained from superior segment of left guinea pig ureter. Frequency and amplitude of smooth muscle spontaneous contraction were measured in Kreb's solution containing or uncontaining 10-4mol/L Glivec before and after the effect of 10-4mol/L carbachol. Then, we detected the expressions of muscarinic receptor subtypes on ICCs of guinea pig ureter. Both c-kit receptor and muscarinic receptor subtypes (M1~M5) on ICCs of guinea pig ureter were been observed with laser scanning confocal microscope after been stained by immunofluorescence.Results:(1) Spontaneous contractions were recorded on isolated smooth muscle strips of guinea pig ureter under certain preload (2.0g) and when 200μL 1.0mol/L KCl solution was added. The frequency and amplitude of spontaneous contraction were (3.4±0.84)/min and (0.105±0.0228)g respectively. (2) When 100μL 0.01mol/L carbachol solution was added, the frequency of spontaneous contraction of ureteral smooth muscle strips increased to (4.9±0.88)/min (p<0.01). While the amplitude was (0.106±0.0210)g, and had no statistical significant difference compared with that before using carbachol (p>0.05).(3) Incubated with Kreb's solution containing 10-4mol/L Glivec 30 minutes for blocking ICCs, then repeat the test in advance, the spontaneous contraction of ureteral smooth muscle strips could not bring out, even in Kreb's solution containing 10-4mol/L carbachol.(4) M2 and M3 of muscarinic receptor subtypes were detected on ICCs of guinea pig ureter by immunofluorescence (double-labeled).Conclusion:Our results indicate that ICCs act as the origin of spontaneous contraction of ureteral smooth muscle and accept regulation of cholinergic nerve through M2 and M3 muscarinic receptor subtypes.