Patients with long-standing diabetes commonly suffer from gastric neuromuscular dysfunction (gastropathy) causing symptoms ranging from postprandial bloating to recurrent vomiting. Autonomic neuropathy is generally believed to be responsible for diabetic gastropathy and the underlying impairments in gastric emptying (gastroparesis) and receptive relaxation, but the specific mechanisms have not been elucidated. Recently, it has been recognized that interstitial cells of Cajal generate electrical pacemaker activity and mediate motor neurotransmission in the stomach. Loss or defects in interstitial cells could contribute to the development of diabetic gastroparesis. Gastric motility was characterized in spontaneously diabetic NOD/LtJ mice by measuring gastric emptying and by monitoring spontaneous and induced electrical activity in circular smooth muscle cells. Interstitial cells of Cajal were studied by Kit immunofluorescence and transmission electron microscopy. Diabetic mice developed delayed gastric emptying, impaired electrical pacemaking, and reduced motor neurotransmission. Interstitial cells of Cajal were greatly reduced in the distal stomach, and the normally close associations between these cells and enteric nerve terminals were infrequent. Our observations suggest that damage to interstitial cells of Cajal may play a key role in the pathogenesis of diabetic gastropathy.

1. Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction.2. Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques.3. Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. 6. These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype.

Morphological studies using immersion or perfusion fixation methods do not reveal the ultrastructure of functioning kidneys with normal circulation. A simple apparatus was developed for freezing the kidneys in vivo without stopping the blood supply, and the ultrastructure of the glomerular capillary loops was examined under different haemodynamic conditions. Mouse kidneys were frozen under normal blood flow conditions; others were frozen in the same way after ligation of the abdominal aorta at a point caudal to the renal arteries. They were then processed for the freeze-substitution or deep-etching method. Good ultrastructural preservation was obtained within about 5 microM depth from the frozen tissue surface. Functioning glomeruli with normal blood flow possessed open capillary lumens, different shapes of foot processes and atypical basement membranes with low density. Moreover, heterogeneity in width between foot processes was identified on the replica membranes. Under the acute conditions used to increase blood supply into the kidneys, the spaces between the flat foot processes became more widely dilated and the basement membrane was seen to be three-layered. The ultrastructure of glomeruli in functioning kidneys has been demonstrated for the first time by this "in vivo cryotechnique."

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Seven known genes were differentially expressed in wildtype and W/W(V) mice. COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug-resistance complex, class 4) were suppressed in both fed and fasted W/W(V) mice. Expression of another five genes was increased in W/W(V) mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL-8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6-5 (encoding phosphorylcholine [PC] T-cell suppressor factor [TsF]). Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state. We conclude that several genes are differentially expressed in the small intestines of W/W(V) mice where the major lesion is loss of pacemaker ICC. Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.