Establishment of pacemaker activity in tissues allotransplanted with interstitial cells of Cajal.

BACKGROUND: Loss or disruption of Kit(+) -interstitial cells of Cajal (ICC) capable of generating pacemaker activity has been implicated in the development of numerous gastrointestinal motility disorders. We sought to develop a model where ICC could be allotransplanted into intestines naturally devoid of these cells. METHODS: Enzymatically dispersed cells from the intestinal tunica muscularis of Kit(+/copGFP) and Kit(V558Δ) /+ gain-of-function mice were allotransplanted into myenteric plexus regions of W/W(V) mutant intestines that lack ICC at the level of the myenteric plexus (ICC-MY) and pacemaker activity. Immunohistochemical analysis fate mapped the development of ICC-MY networks and intracellular microelectrode recordings provided evidence for the development of functional pacemaker activity. KEY RESULTS: Kit(+) -ICC developed into distinct networks at the level of the myenteric plexus in organotypic cultures over 28 days and displayed robust rhythmic pacemaker activity. CONCLUSIONS & INFERENCES: This study demonstrates the feasibility of allotransplantation of ICC into the myenteric region of the small intestine and the establishment of functional pacemaker activity into tissues normally devoid of ICC-MY and slow waves, thus providing a possible basis for the therapeutic treatment of patients where ICC networks have been disrupted due to a variety of pathophysiological conditions.

Relationship between enteric neurons and interstitial cells in the primate gastrointestinal tract.

BACKGROUND: Morphological studies have revealed a close anatomical relationship between enteric nerve terminals and intramuscular ICC (ICC-IM) which supports a role for ICC-IM as intermediaries in enteric motor neurotransmission. Recently, a second type of interstitial cell previously described as 'fibroblast-like' but can now be identified by platelet-derived growth factor receptor-α expression, has also been implicated in enteric neurotransmission in rodents. The present study was performed to determine if enteric nerve fibers form close anatomical relationships with ICC and PDGFRα(+) cells throughout the primate GI tract. METHODS: Immunohistochemical experiments and confocal microscopy were performed to examine the relationship between excitatory and inhibitory motor neurons, ICC and PDGFRα(+) cells throughout the monkey GI tract. KEY RESULTS: The pan neuronal marker. Protein gene product 9.5 (PGP9.5) was used to label all enteric neurons and substance-P (sub-P) and neuronal nitric oxide synthase (nNOS) to label excitatory and inhibitory neurons, respectively. Double labeling with Kit revealed that both classes of nerve fibers were closely apposed with ICC-IM in the stomach, small intestine and colon (taenia and inter-taenia regions), but not with ICC at the level of the myenteric plexus (ICC-MY). Varicose enteric nerve fibers were closely associated with ICC-IM for distances up to 250 µm. Both excitatory and inhibitory nerve fibers were also closely apposed to PDGFRα(+) cells throughout the primate GI tract. CONCLUSIONS & INFERENCES: The close anatomical relationship between enteric nerve fibers and ICC-IM and PDGFRα(+) cells throughout the GI tract of the Cynomolgus monkey provides morphological evidence that these two classes of interstitial cells may provide a similar physiological function in primates as has been attributed in rodent animal models.

Interstitial cells of Cajal in the cynomolgus monkey rectoanal region and their relationship to sympathetic and nitrergic nerves.

The morphology of interstitial cells of Cajal (ICC) in the circular muscle layer of the cynomolgus monkey internal anal sphincter (IAS) and rectum and their relationship to sympathetic and nitrergic nerves were compared by dual-labeling immunohistochemistry. Contractile studies confirmed that nitrergic nerves participate in neural inhibition in both regions whereas sympathetic nerves serve as excitatory motor nerves only in the IAS. Muscle bundles extended from myenteric to submucosal edge in rectum but in the IAS bundles were further divided into "minibundles" each surrounded by connective tissue. Dual labeling of KIT and smooth muscle myosin revealed KIT-positive stellate-shaped ICC (ICC-IAS) within each minibundle. In the rectum intramuscular ICC (ICC-IM) were spindle shaped whereas stellate-shaped ICC were located at the myenteric surface (ICC-MY). ICC were absent from both the myenteric and submucosal surfaces of the IAS. Nitrergic nerves (identified with anti-neuronal nitric oxide synthase antibodies or NADPH diaphorase activity) and sympathetic nerves (identified with anti-tyrosine hydroxylase antibody) each formed a plexus at the myenteric surface of the rectum but not the IAS. Intramuscular neuronal nitric oxide synthase- and tyrosine hydroxylase-positive fibers were present in both regions but were only closely associated with ICC-IM in rectum. Minimal association was also noted between ICC-IAS and cells expressing the nonspecific neuronal marker PGP9.5. In conclusion, the morphology of rectal ICC-IM and ICC-MY is similar to that described elsewhere in the gastrointestinal tract whereas ICC-IAS are unique. The distribution of stellate-shaped ICC-IAS throughout the musculature and their absence from both the myenteric and submucosal surfaces suggest that ICC-IAS may serve as pacemaker cells in this muscle whereas their limited relationship to nerves suggests that they are not involved in neuromuscular transmission. Additionally, the presence of numerous minibundles, each containing both ICC-IAS and nerves, suggests that this muscle functions as a multiunit type muscle.

Comparison of mechanical and electrical activity and interstitial cells of Cajal in urinary bladders from wild-type and W/Wv mice.

BACKGROUND AND PURPOSE: W/W(v) and wild-type murine bladders were studied to determine whether the W/W(v) phenotype, which causes a reduction in, but not abolition of, tyrosine kinase activity, is a useful tool to study the function of bladder interstitial cells of Cajal (ICC). EXPERIMENTAL APPROACH: Immunohistochemistry, tension recordings and microelectrode recordings of membrane potential were performed on wild-type and mutant bladders. KEY RESULTS: Wild-type and W/W(v) detrusors contained c-Kit- and vimentin-immunopositive cells in comparable quantities, distribution and morphology. Electrical field stimulation evoked tetrodotoxin-sensitive contractions in wild-type and W/W(v) detrusor strips. Atropine reduced wild-type responses by 50% whereas a 25% reduction occurred in W/W(v) strips. The atropine-insensitive component was blocked by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both tissue types. Wild-type and W/W(v) detrusors had similar resting membrane potentials of -48 mV. Spontaneous electrical activity in both tissue types comprised action potentials and unitary potentials. Action potentials were nifedipine-sensitive whereas unitary potentials were not. Excitatory junction potentials were evoked by single pulses in both tissues. These were reduced by atropine in wild-type tissues but not in W/W(v) preparations. The atropine-insensitive component was abolished by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both preparations. CONCLUSIONS AND IMPLICATIONS: Bladders from W/W(v) mice contain c-Kit- and vimentin-immunopositive ICC. There are similarities in the electrical and contractile properties of W/W(v) and wild-type detrusors. However, significant differences were found in the pharmacology of the responses to neurogenic stimulation with an apparent up-regulation of the purinergic component. These findings indicate that the W/W(v) strain may not be the best model to study ICC function in the bladder.

Properties of unitary potentials generated by intramuscular interstitial cells of Cajal in the murine and guinea-pig gastric fundus.

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse and guinea-pig gastric fundus. These preparations displayed an ongoing discharge of membrane noise (unitary potentials), similar to that recorded from similar preparations made from the circular layer of the antrum. Bundles of muscle from the fundus of W/W(V) mice, which lack intramuscular interstitial cells of Cajal (ICC(IM)) lacked the discharge of membrane noise observed in wild-type tissues. When the membrane potential was changed by passing depolarizing or hyperpolarizing current pulses, the discharge of membrane noise was little changed. The membrane noise was unaffected by adding chloride channel blockers; however, agents which buffered the internal concentration of calcium ions reduced the discharge of membrane noise. Treatment of tissues with CCCP, which interferes with the uptake of calcium ions by mitochondria, also reduced the membrane noise and caused membrane hyperpolarization. Similar observations were made on bundles of tissue isolated from the circular layer of the guinea pig antrum. Together the observations indicate that membrane noise is generated by a pathway located in ICC(IM). The properties of this pathway appear to vary dramatically within a given organ. The lack of voltage sensitivity of the discharge of membrane noise in the fundus provides a possible explanation for the lack of rhythmic electrical activity in this region of the stomach.

Involvement of intramuscular interstitial cells in nitrergic inhibition in the mouse gastric antrum.

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse gastric antrum and the responses evoked by stimulating intrinsic nerve fibres were examined. Transmural nerve stimulation evoked a fast inhibitory junction potential (fast-IJP) which was followed initially by a smaller amplitude long lasting inhibitory junction potential (slow-IJP) and a period of excitation. The excitatory component of the response was abolished by atropine, suggesting that it resulted from the release of acetylcholine and activation of muscarinic receptors. Fast-IJPs were selectively reduced in amplitude by apamin and slow-IJPs were abolished by N(omega)-nitro-L-arginine. Slow-IJPs were associated with a drop in membrane noise, suggesting that inhibition resulted from a reduced discharge of unitary potentials by intramuscular interstitial cells of Cajal (ICC(IM)). The chloride channel blocker, anthracene-9-carboxylic acid, reduced the discharge of membrane noise in a manner similar to that detected during the slow-IJP. When recordings were made from the antrum of W/W(V) mice, which lack ICC(IM), the cholinergic and nitrergic components were absent, with only fast-IJPs being detected. The observations suggest that neurally released nitric oxide selectively targets ICC(IM) causing a hyperpolarization by suppressing the discharge of unitary potentials.

Ultrastructural analysis of the transdifferentiation of smooth muscle to skeletal muscle in the murine esophagus.

The ultrastructure of the mouse esophagus at the level of the diaphragm was studied from embryo day 17 to adult. The transdifferentiation of smooth muscle into skeletal muscle was categorized into seven ultrastructural stages: during phase I normal smooth muscle myogenesis was observed. In phase II subpopulations of cells changed into aggregates of myoblast-like cells. At the center of these cell aggregates, phase III cells appeared that contained condensed myofilaments. Dense bodies and dense bands appeared enlarged by the accumulation of thin filaments. In phase IV the condensed myofilaments organized into sarcomere pretemplate structures. The dense bodies and dense bands formed rudimentary Z-lines. In phase V the sarcomere templates appeared as more defined structures and began to align. An elaborate perinuclear region appeared. During phase VI, skeletal muscle sarcomeres were apparent and myofilaments were arranged in a typical hexagonal array. Phase VII skeletal muscle fibers were unique with sarcomeric bifurcations and anastomoses between adjacent myofibrils. Non-contractile organelles were less organized in these cells than in skeletal muscles such as rectus and vastus lateralis muscles. During the transdifferentiation process, other cell types remained unchanged, except the number of interstitial cells of Cajal became reduced. Immunocytochemical studies with antibodies against smooth and skeletal muscle myosin were also performed during the process of transdifferentiation. An osmium tetroxide/potassium ferricyanide en bloc mordant enabled the use of ultrathin Unicryl sections for immunocytochemistry. Cells exhibited smooth muscle myosin-like immunoreactivity from the smooth muscle stage through the condensed myofilament stage. Cells were immunopositive for skeletal muscle myosin before the formation of sarcomere templates, during the condensed stage, and after development of mature skeletal muscle cells. We also observed a hybrid muscle cell with properties of both smooth and skeletal muscle cells.