InsP3R-associated cGMP kinase substrate (IRAG) is essential for nitric oxide-induced inhibition of calcium signaling in human colonic smooth muscle.
Nitric oxide (NO)-mediated relaxation of colonic smooth muscle is crucial for the maintenance of human gut function. The molecular mechanisms of NO-dependent smooth muscle relaxation involve cyclic GMP-mediated inhibition of store-dependent calcium signaling. Recently, IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate) has been characterized as a novel target molecule of cGMP-dependent protein kinase (cGKI) mediating NO-/cGMP-dependent inhibition of inositol 1,4,5-trisphosphate (InsP(3))-dependent calcium release in transfected COS cells. The aim of the present study was to characterize IRAG expression and its functional role in NO-dependent signaling in human colonic smooth muscle. Reverse transcriptase-PCR revealed IRAG mRNA expression in human colon, rectum, and cultured colonic smooth muscle cells. In cultured human colonic smooth muscle cells, bradykinin (BK) elicited InsP(3)-dependent calcium transients that were repeatable and independent of extracellular calcium. The NO donor sodium nitroprusside and the specific cGK activator 8-(4-chlorophenylthio)guanosine-3',5'-cyclic-monophosphate (8-pCPT-cGMP) significantly inhibited BK-induced increase in intracellular calcium. Cells transfected with antisense oligonucleotides raised against IRAG (IRAG-AS) showed strongly decreased IRAG protein expression. In these cells, sodium nitroprusside and 8-pCPT-cGMP both failed to modulate BK-induced calcium transients. Thus, endogenous IRAG appears to be essentially involved in the NO/cGK-dependent inhibition of InsP(3)-dependent Ca(2+)-signaling in colonic smooth muscle.
Fritsch
Ralph M
RM
Department of Internal Medicine II, Technische Universitat München, Ismaninger Strasse 22, 81675 Munich, Germany. ralph.fritsch@lrz.tum.de
Saur
Dieter
D
Kurjak
Manfred
M
Oesterle
Daniela
D
Schlossmann
Jens
J
Geiselhöringer
Angela
A
Hofmann
Franz
F
Allescher
Hans-Dieter
HD
D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003106:Colon / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry* / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012007:Rectum / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
C485784:IRAG1 protein, human; D008565:Membrane Proteins; D020030:Nitric Oxide Donors; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D012333:RNA, Messenger; D013873:Thionucleotides; D009599:Nitroprusside; D009569:Nitric Oxide; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D006152:Cyclic GMP; D001920:Bradykinin; D002118:Calcium