Anderie I, Blum R, Haase W, Grinstein S, Thévenod F
II, Department of Physiology, University of the Saarland, Homburg/Saar, Germany.
Biochem Biophys Res Commun. 1998 May 19;246(2):330-6. doi: 10.1006/bbrc.1998.8611.
We previously characterized a Na+/H+ exchange activity in rat pancreatic zymogen granules [Anderie, I., and Thévenod, F. (1996) J. Membrane Biol, 152, 195-205]. Here we have identified the Na+/H+ exchanger (NHE) isoforms present in zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited zymogen granule Na+/H+ exchange in a concentration dependent manner, maximally to 53 +/- 5% of controls at 100nM. The remaining Na+/H+ exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC50 approximately 25 microM) or benzamil (EC50 approximately 100 microM). Amiloride inhibited weakly suggesting that "amiloride-resistant" and "amiloride-sensitive" NHE are expressed in zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in zymogen granule membranes was confirmed by immunoblots of zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in zymogen granule membranes of rat exocrine pancreas.
我们之前已对大鼠胰腺酶原颗粒中的Na⁺/H⁺交换活性进行了表征[安德里，I.，和特韦诺德，F.（1996年）《膜生物学杂志》，152，195 - 205]。在此，我们通过使用Na⁺/H⁺交换抑制剂的功能研究，鉴定了存在于酶原颗粒中的Na⁺/H⁺交换体（NHE）亚型。NHE1特异性阻滞剂HOE 694[3 - （甲基磺酰基 - 4 - 哌啶基 - 苯甲酰基） - 胍甲磺酸盐]以浓度依赖性方式抑制酶原颗粒的Na⁺/H⁺交换，在100 nM时最大抑制至对照的53±5%。其余的Na⁺/H⁺交换活性可被EIPA[5 - （N - 乙基 - N - 异丙基）氨氯吡咪]（EC50约为25 μM）或苄甲氯噻嗪（EC50约为100 μM）抑制。氨氯吡咪抑制作用较弱，表明“氨氯吡咪抗性”和“氨氯吡咪敏感性”NHE在酶原颗粒中均有表达。通过逆转录聚合酶链反应（RT - PCR）在大鼠胰腺组织和大鼠胰腺腺泡细胞系AR4 - 2J中检测到了编码NHE1和NHE4特异性跨膜结构域的cDNA序列。通过酶原颗粒膜的免疫印迹以及用多克隆NHE1和NHE4抗体对纯化的大鼠胰腺酶原颗粒进行预包埋免疫金标记，证实了NHE1和NHE4存在于酶原颗粒膜中。因此，我们提出NHE1和NHE4在大鼠外分泌胰腺的酶原颗粒膜中表达。
