Broere N, Chen M, Cinar A, Singh A K, Hillesheim J, Riederer B, Lünnemann M, Rottinghaus I, Krabbenhöft A, Engelhardt R, Rausch B, Weinman E J, Donowitz M, Hubbard A, Kocher O, de Jonge H R, Hogema B M, Seidler U
Department of Biochemistry, Erasmus University Medical Center, Rotterdam, The Netherlands.
Pflugers Arch. 2009 Mar;457(5):1079-91. doi: 10.1007/s00424-008-0579-1. Epub 2008 Aug 29.
We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.
我们研究了钠氢交换调节因子1（NHERF1）在小鼠肠道盐和水吸收、刷状缘膜（BBM）形态以及NHE3 mRNA表达、蛋白质丰度和转运活性方面的作用。NHERF1基因缺陷小鼠在体内空肠液体吸收减少，在体外分离的空肠和结肠黏膜中钠吸收减弱，但回肠黏膜未出现这种情况。然而，cAMP介导的对这两个参数的抑制作用保持完整。在结肠表面细胞中，酸激活的NHE3转运速率降低，但其受cAMP和cGMP的抑制作用正常。对NHE3的免疫检测显示，NHE3在NHERF1基因敲除小鼠的BBM中定位正常，但通过蛋白质印迹法测定，从小肠和大肠分离的BBM中NHE3丰度显著降低。此外，NHERF1基因敲除小鼠近端结肠的微绒毛明显变短，但小肠中的微绒毛没有变化。另外敲除PDZK1（NHERF3），它是NHERF衔接蛋白家族的另一个成员，能与NHE3和NHERF1结合，这进一步降低了基础NHE3活性，并导致cAMP介导的NHE3抑制作用完全丧失。cAMP激活的交换蛋白（EPAC）的激活剂对体内空肠液体吸收没有影响，但在体外能轻微抑制结肠表面细胞中的NHE3活性。总之，NHERF1对肠道盐吸收、NHE3转运速率和NHE3膜丰度具有节段特异性作用，而不影响mRNA水平。然而，与PDZK1不同，NHERF1不是环核苷酸调节NHE3所必需的。
