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J. Biol. Chem., Vol. 282, Issue 34, 25141-25151, August 24, 2007

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Tissue-specific Regulation of Sodium/Proton Exchanger Isoform 3 Activity in Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1) Null Mice

cAMP INHIBITION IS DIFFERENTIALLY DEPENDENT ON NHERF1 AND EXCHANGE PROTEIN DIRECTLY ACTIVATED BY cAMP IN ILEUM VERSUS PROXIMAL TUBULE^*

Rakhilya Murtazina, Olga Kovbasnjuk, Nicholas C. Zachos, Xuhang Li, Yueping Chen, Ann Hubbard^¶, Boris M. Hogema^||, Deborah Steplock^**, Ursula Seidler, Kazi M. Hoque, Chung Ming Tse, Hugo R. De Jonge^||, Edward J. Weinman^**, and M. Donowitz¹

From the Departments of Medicine, Physiology, and ^¶Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, ^||Department of Biochemistry, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands, ^**Department of Medicine, University of Maryland School of Medicine and Medical Staff, Department of Veteran Affairs, Baltimore, Maryland 21201, and Department of Medicine, Hannover University School of Medicine, 2 D-30419 Hannover, Germany

The multi-PDZ domain containing protein Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1) binds to Na⁺/H⁺ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1^-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1^-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1^-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1^-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

Received for publication, March 5, 2007 , and in revised form, May 31, 2007.

^* This work was supported in part by the NIDDK, National Institutes of Health Grants RO1-DK26523, RO1-DK61765, PO1-DK44484, PO1-DK72084, R24-DK64388 (to The Hopkins Basic Research Digestive Diseases Development Core Center) and by the Hopkins Center for Epithelial Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5, references, and discussion.

¹ To whom correspondence should be addressed: GI Division, Dept. of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Ave. 925 Ross Research Bldg., Baltimore, MD 21205. Tel.: 410-955-9675; Fax: 410-955-9677; E-mail: mdonowit{at}jhmi.edu.

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