Abstract
MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.
Highlights
Maintenance of intestinal homeostasis is closely linked to secretory mechanisms controlling fluid and electrolyte secretion, regulated by coordinated actions of a complex network of transporters and ion channels located in the apical and basolateral membranes of intestinal epithelial cells (IECs)
We further investigated the cellular localization of MRP4 expressed in rat colonic mucosa by immunohistochemistry and found MRP4specific staining predominantly associated with the apical membrane, indicating high expression of this cGMP transporter on the apical membrane of rat colonocytes, with very minor staining associated with the basolateral membrane detectable, a pattern similar to that described recently in human and mouse colonic mucosa (Harrington et al, 2014) and previously reported for human colonic T84 and HT29-CL19A epithelial cells (Li et al, 2007) (Fig. 3)
We provide evidence for a direct role of MRP4 as a key modulator of linaclotide pharmacology in the rat colon: potentiation of electrolyte secretion, augmentation of tissue cGMP accumulation, and inhibition of cGMP efflux selectively from the apical membrane of polarized IECs
Summary
Maintenance of intestinal homeostasis is closely linked to secretory mechanisms controlling fluid and electrolyte secretion, regulated by coordinated actions of a complex network of transporters and ion channels located in the apical and basolateral membranes of intestinal epithelial cells (IECs). The movement of Cl2 ions across the apical membrane of IECs generates an electrogenic ion current that drives transepithelial sodium flux. This process is further associated with inhibition of NHE3mediated sodium reabsorption, thereby establishing an osmotic gradient for concomitant movement of water into the luminal compartment. The main conductive pathway for Cl2 ions across the apical membrane of IECs is the anion-selective CFTR channel.
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