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The distribution of the neurokinin type 1 receptor (NK1r) in human intestine, mapped in a few immunohistochemical investigations in the antrum and the duodenum, is comparable to that widely studied in rodents. Importantly, despite pharmacological evidence of their presence in mammalian intestinal muscle, their immunohistochemical visualization in smooth muscle cells remains to be determined in human digestive tract. In the present work, we studied the distribution of NK1r in the human colon, with a particular view to visualize their expression in muscle cells. With this aim, part of colonic segments were incubated with nicardipine and TTX in order to induce accumulation of the NK1r on cell membrane. NK1r were visualized by using immunohistochemistry combined with fluorescence and confocal microscopy. Without incubation, NK1r-IR was clearly observed on the membrane and the cytoplasm of myenteric and submucous neurons and interstitial cells of Cajal, but could not be clearly determined in the longitudinal and circular muscle. NK1r-IR-expressing neurons and interstitial cells were closely surrounded by substance P (SP) immunoreactive nerves. Incubation of colonic segments with nicardipine and TTX at 4 degrees C for 1 h with SP allowed to reveal a strong NK1r-IR at the surface of muscle cells. Incubation with SP (10(-6) M) at 37 degrees C for 1 min induced a relocation of NK1r-IR into the cytoplasm of muscle. This is interpreted as an internalization of NK1r induced by the binding of SP on muscular NK1r. The present data contribute to emphasize the role of NK1r in tachykinin-mediated neuronal processes regulating intestinal motility.

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between -100 and -40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I(-) > SCN(-) > Br(-) > Cl(-) > NO(3)(-) > F(-)>> SO(4)(2-) approximately gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca(2+) concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5'-O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5'-O-(2-thiodiphosphate) reactivated the channel.

The effects of hypothalamic microinfusions of cholecystokinin octapeptide and its antagonist L364,718 on cecocolonic myoelectrical activity were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the cecum and proximal colon and cannulas placed bilaterally in either the ventromedial or lateral hypothalamus. In fasted rats, microinfusion of cholecystokinin octapeptide (10 ng/kg) into the ventromedial hypothalamus increased the spike-burst frequency of the cecum and the colon by 45.6% and 43.7%, respectively, during the 30-minute period after treatment. The injection of cholecystokinin octapeptide (10 ng/kg) into the lateral hypothalamus had no effect on either cecal or colonic motility. Feeding increased the frequency of cecal and colonic spike bursts by 52.1% and 50.1% for 30 minutes postprandially. When infused bilaterally into the ventromedial hypothalamus 10 minutes before feeding, L364,718 (1 or 5 micrograms/kg) abolished the increase of the frequency of cecal and colonic contractions induced by the meal. Infused into the lateral hypothalamus at similar dosages, L364,718 had no effect on the postprandial enhancement of cecocolonic motility. Increase of cecocolonic spike-burst frequency induced by feeding or by cholecystokinin octapeptide injected into the ventromedial hypothalamus was abolished by previous intracerebroventricular but not intraperitoneal administration of atropine (1 microgram) and 4-diphenylacetoxy-N-methylpiperidine (1 microgram), a selective muscarinic M2-receptor antagonist. In contrast, pirenzepine (1 microgram, intracerebroventricularly) did not significantly reduce the meal- or cholecystokin octapeptide-induced increase in cecal and colonic motility. These results suggest that, in rats, (a) cholecystokinin octapeptide is involved in the generation of the cecocolonic motor response to a meal and these effects are mediated through cholecystokinin octapeptide receptors located in the ventromedial hypothalamic nuclei, and (b) these postprandial colonic motor changes involve central cholinergic activation through muscarinic M2 receptors.