Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K ؉ -Cl ؊ cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na ؉ ,K ؉ -ATPase ␣1-subunit (␣1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabainsensitive K ؉ -dependent ATP-hydrolyzing activity (Na ؉ ,K ؉ -
ATPase activity) was significantly inhibited by a KCC inhibitor (R-(؉)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in associ-ation of KCC3a with endogenous ␣1NaK, and it recruited ␣1NaK in lipid rafts, accompanying increases of Na ؉ ,K ؉ -ATPase activity and ouabain-sensitive Na ؉ transport activity that were suppressed by DIOA, whereas the total expression level of ␣1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with ␣1NaK. In conclusion, KCC3a forms a functional complex with ␣1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates ␣1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.
Background
Because M1 muscarinic receptors are expressed by enteric neurons, we investigated whether positive allosteric modulators of these receptors (M1PAMs) would enhance colorectal propulsion and defecation in dogs, mice, and rats.
Methods
The potencies of the M1PAMs, T662 or T523, were investigated using M1 receptor‐expressing CHO cells. Effectiveness of M1PAMs on defecation was investigated by oral administration in mice and rats, by recording propulsive contractions in anaesthetized rats and by recording high amplitude propagating contractions in dogs.
Key Results
PAM EC50 values in M1 receptor‐expressing CHO cells were 0.7‐1.8 nmol/L for T662 and 8‐10 nmol/L for T523. The compounds had 1000‐fold lower potencies as agonists. In anesthetized rats, both compounds elicited propulsive colorectal contractions, and in dogs, mice, and rats, oral administration increased fecal output. No adverse effects were observed in conscious animals. M1PAMs triggered propagated high amplitude contractions and caused defecation in dogs. Nerve‐mediated contractions were enhanced in the isolated mouse colon. M1PAMs were equi‐effective in rats with or without the pelvic nerves being severed. In two models of constipation in mice, opiate‐induced constipation and constipation of aging, defecation was induced and constipation was reversed.
Conclusion and Inferences
M1PAMs act at targets sites in the colorectum to enhance colorectal propulsion. They are effective across species, and they reverse experimentally induced constipation. Previous studies have shown that they are safe in human. Because they provide an enhancement of physiological control rather than being direct agonists, they are predicted to provide effective treatment for constipation.
It is known that specific alteration of rhythm in temperature (SART) stress produces somatic pain. However, it remains to be investigated whether SART stress induces visceral pain. In this study, we investigated the visceral hypersensitivity in the SART stress model by pharmacological tools and heterotopical nociception. Four-week-old Sprague-Dawley rats were exposed to repeated cold stress. Visceral pain was measured by visceromotor response to colorectal distension, and the effects of alosetron and duloxetine on visceral pain were investigated in SART rats. Heterotopical nociception was given by capsaicin injection into the left forepaw to induce diffuse noxious inhibitory controls (DNIC). SART stress induced visceral hypersensitivity that was sustained at minimum for one week. In pharmacological analysis, alosetron and duloxetine improved SART stress-induced visceral hypersensitivity. Heterotopical nociception induced DNIC in normal conditions, but was disrupted in SART rats. On the other hand, RMCP-II mRNA in distal colon was not affected by SART stress. In conclusion, SART rats exhibit several features of visceral pain in IBS, and may be a useful model for investigating the central modification of pain control in IBS.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.