title: MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.
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.
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Boris
authors.initials: B
authors.lastname: Tchernychev
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Pei
authors.initials: P
authors.lastname: Ge
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Marco M
authors.initials: MM
authors.lastname: Kessler
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Robert M
authors.initials: RM
authors.lastname: Solinga
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Derek
authors.initials: D
authors.lastname: Wachtel
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Jenny V
authors.initials: JV
authors.lastname: Tobin
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Sara R
authors.initials: SR
authors.lastname: Thomas
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Craig E
authors.initials: CE
authors.lastname: Lunte
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Angelika
authors.initials: A
authors.lastname: Fretzen
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hannig
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Bryant
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Caroline B
authors.initials: CB
authors.lastname: Kurtz
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.).
authors.forename: Mark G
authors.initials: MG
authors.lastname: Currie
authors.affiliation: Ironwood Pharmaceuticals, Cambridge, Massachusetts (B.T., P.G., M.M.K., R.M.S., D.W., J.V.T., A.F., G.H., A.P.B., C.B.K., M.G.C., I.S.-S.); and Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas (S.R.T., C.E.L.) isilos-santiago@ironwoodpharma.com.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Silos-Santiago
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006152:Cyclic GMP / Q000378:metabolism*; D004573:Electrolytes / Q000378:metabolism*; D055724:Electrophysiological Phenomena / Q000187:drug effects; D005260:Female; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000494:pharmacology*; D011422:Propionates / Q000494:pharmacology*; D011804:Quinolines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D006152:Cyclic GMP; D004573:Electrolytes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010455:Peptides; D011422:Propionates; D011804:Quinolines; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; C489542:Abcc4 protein, rat; C059141:verlukast; C523483:linaclotide