Fritsch Ralph M, Saur Dieter, Kurjak Manfred, Oesterle Daniela, Schlossmann Jens, Geiselhöringer Angela, Hofmann Franz, Allescher Hans-Dieter
Department of Internal Medicine II, Technische Universitat München, Ismaninger Strasse 22, 81675 Munich, Germany.
J Biol Chem. 2004 Mar 26;279(13):12551-9. doi: 10.1074/jbc.M313365200. Epub 2004 Jan 18.
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.
一氧化氮（NO）介导的结肠平滑肌舒张对于维持人体肠道功能至关重要。NO依赖性平滑肌舒张的分子机制涉及环鸟苷酸（cGMP）介导的对储存依赖性钙信号的抑制。最近，肌醇1,4,5-三磷酸受体相关cGMP激酶底物（IRAG）已被鉴定为cGMP依赖性蛋白激酶（cGKI）的一种新型靶分子，它介导在转染的COS细胞中NO/cGMP依赖性抑制肌醇1,4,5-三磷酸（InsP(3)）依赖性钙释放。本研究的目的是鉴定IRAG在人结肠平滑肌NO依赖性信号传导中的表达及其功能作用。逆转录聚合酶链反应（RT-PCR）显示IRAG mRNA在人结肠、直肠和培养的结肠平滑肌细胞中表达。在培养的人结肠平滑肌细胞中，缓激肽（BK）引发了可重复且独立于细胞外钙的InsP(3)依赖性钙瞬变。NO供体硝普钠和特异性cGK激活剂8-（4-氯苯硫基）鸟苷-3',5'-环一磷酸（8-pCPT-cGMP）显著抑制BK诱导的细胞内钙增加。用针对IRAG的反义寡核苷酸转染的细胞（IRAG-AS）显示IRAG蛋白表达大幅下降。在这些细胞中，硝普钠和8-pCPT-cGMP均未能调节BK诱导的钙瞬变。因此，内源性IRAG似乎在结肠平滑肌中NO/cGK依赖性抑制InsP(3)依赖性Ca(2+)信号传导中起重要作用。
