ABSTRACT
In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.
Subject(s)
Action Potentials/physiology , Biological Clocks/physiology , Intestine, Small/physiology , Myocytes, Smooth Muscle/physiology , Sodium-Potassium-Chloride Symporters/metabolism , Animals , Cells, Cultured , In Situ Hybridization , Mice , Mice, Transgenic , Solute Carrier Family 12, Member 2ABSTRACT
BACKGROUND & AIMS: In patients with chronic intestinal pseudo-obstruction, intestinal motility is disturbed by either nervous or myogenic aberrations. The cause of the myogenic form is unknown, but it is likely to originate in the contractile apparatus of the smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Experimental data indicate a role for smoothelins in smooth muscle contraction. A smoothelin-deficient mouse model may help to establish the role of smoothelin-A in intestinal contraction and provide a model for myogenic chronic intestinal pseudo-obstruction. METHODS: We used gene targeting to investigate the function of smoothelin-A in intestinal tissues. By deletion of exons 18, 19, and 20 from the smoothelin gene, the expression of both smoothelin isoforms was disrupted. The effects of the deficiency were evaluated by pathologic and physiologic analyses. RESULTS: In smoothelin-A/B knockout mice, the intestine was fragile and less flexible compared with wild-type littermates. The circular and longitudinal muscle layers of the intestine were hypertrophic. Deficiency of smoothelin-A led to irregular slow wave patterns and impaired contraction of intestinal smooth muscle, leading to hampered transport in vivo. This caused obstructions that provoked intestinal diverticulosis and occasionally intestinal rupture. CONCLUSIONS: Smoothelin-A is essential for functional contractility of intestinal smooth muscle. Hampered intestinal transit in smoothelin-A/B knockout mice causes obstruction, starvation, and, ultimately, premature death. The pathology of mice lacking smoothelin-A is reminiscent of that seen in patients with chronic intestinal pseudo-obstruction.
Subject(s)
Cytoskeletal Proteins/physiology , Intestinal Pseudo-Obstruction/physiopathology , Intestines/physiology , Muscle Contraction/physiology , Muscle Proteins/physiology , Muscle, Smooth/physiology , Animals , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Disease Models, Animal , Diverticulum/genetics , Diverticulum/pathology , Diverticulum/physiopathology , Female , Gene Expression , Immunohistochemistry , Intestinal Pseudo-Obstruction/genetics , Intestinal Pseudo-Obstruction/pathology , Intestines/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Smooth/pathology , Phenotype , PregnancyABSTRACT
PURPOSE: The chemical coding of intrinsic choroidal neurons (ICNs) has features in common with extrinsic fibers (e.g., from the pterygopalatine ganglion) making it impossible to assess whether a neuronal nitric oxide synthase (nNOS)/vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fiber is of intrinsic or extrinsic origin. Neurobiotin injections into single neurons allow the visualization of projections of these cells and the determination of the origin of target innervation. Thus, this technique was used in the present study to help characterize the organization of the ICN in the human eye. METHODS: ICNs were visualized with the fluorescent vital dye 4-Di-2-ASP. Electrophysiological properties were determined by means of intracellular recordings. The impaled neurons were iontophoretically filled with neurobiotin. After fixation, immunohistochemistry for neuronal nitric oxide synthase (nNOS), alpha-smooth muscle actin, and calcitonin gene-related peptide (CGRP) was conducted. RESULTS: ICN processes were traced over distances of up to 2.612 micro m. They were found in the immediate vicinity of other nNOS-positive or -negative ICNs and were also found apposed to smooth muscle fibers (vascular and stromal nonvascular). CGRP-positive fibers forming boutons were observed closely associated with ICNs. Electrophysiological recording showed phasic firing without slow afterhyperpolarization, no spontaneous activity, an input resistance of 136 +/-73 MOmega, and a membrane time constant of 7 +/- 1 ms. CONCLUSIONS: Apart from the first functional characterization of ICNs, this study provided more precise evidence of reciprocal ICN-to-ICN contacts and innervation of both choroidal nonvascular and vascular smooth muscle. The presented technique offers promising perspectives to further investigate the function of ICNs in ocular homeostasis.
