Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO 3− Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct

Abstract

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO 3 ؊ secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO 3 ؊ secretion, the role of CFTR in HCO 3 ؊ salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO 3 ؊ salvage mechanism Na ؉ /H ؉ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote ⌬F508 mice. Accordingly, luminal Na ؉ -dependent and HOE694-sensitive recovery from an acid load was reduced by 60% in ducts of ⌬F508 mice. CFTR and NHE3 were coimmunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of ⌬F508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO 3 ؊ homeostasis by regulating both pancreatic ductal HCO 3 ؊ secretory and salvage mechanisms.Fluid secretion and the control of the ionic composition of biological fluids are essential for the function of many secretory epithelia, including the respiratory, digestive, and reproductive systems. HCO 3 Ϫ is an important constituent of secreted fluids by virtue of its function as the biological pH buffer and its effect on the solubility of proteins and ions in biological fluids. Accordingly, the mechanisms underlying HCO 3 Ϫ homeostasis at rest and during stimulation have attracted much attention in recent years. The importance of HCO 3 Ϫ homeostasis is evident from the marked reduction in Cl Ϫ -and HCO 3 Ϫ

Disruption of IFT results in both exocrine and endocrine abnormalities in the pancreas of Tg737 mutant mice

“…This would be similar to the pathology seen in the pancreas of CFTR mutant mice where defects in chloride transport in the duct epithelium results in lack of fluid secretion and premature activation of pancreatic digestive enzymes. [85][86][87] Alternatively, there may be an unidentified signaling pathway between the ducts and acini required for acinar cell survival, maintenance, or regulation of zymogen granule release.…”

Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

“…Immunostaining of frozen sections was performed as previously reported (8). Briefly, the sections were fixed and permeabilized by incubation in cold methanol for 10 min at Ϫ20°C.…”

“…RT-PCR-RT-PCR analysis was performed using rat pancreatic tissues and isolated pancreatic duct cells as reported previously (8). The primer sequences used for this study were as follows: 1) rSAP102, sense (GTA CCC GGC AAG AAC ACC CCA AAA CTC AAC), antisense (CCG CAC CAC CAA CCG CAC CAC A), PCR product 432 bp; 2) rSAP97, sense (GTC GTC CTG CCC TCC ACA CCA CA), antisense (CCT TCC GCC TTT TCA CAT ATA ATC GCA CTA), PCR product 376 bp; 3) rGKAP, sense (GAG GCC GTT CAA AGG TCC GTG TGC), antisense (GGG GCC CTT CGC TCC TTC TTG TCA), PCR product 395 bp; 4) rPSD-93, sense (AAG ACT TCC CTG CCG CCC ATC C), antisense (TGC CCC GCC CCC TTC AGT G), PCR product 530 bp; 5) rPSD-95, sense (CCC CAG CCT CCC TTC CCA CCC TTC TTA TTT), antisense (ATG GGG AGT TAT GAT GGG GCA GGG GTG AC), PCR product 373 bp; 6) rShank1, sense (TCT TAT AGG GGG AGC TTT GGA CAC AGG A), antisense (GAC GGG GGA CAG CAG CAT CAC AGG), PCR product 366 bp; 7) rShank2, sense (CCC CGC AGC CGC TCT CCC TCA CT), antisense (CCG CCC TCG CCG ATG CTC AGA ACT T), PCR product 278 bp; 8) rS-SCAM, sense (ACA GGC AGC CAC AGG ATT TTG ATT ATT T), antisense (CAG GCG TGG GGT TCG TCG TGT CTT TAG), PCR product 402 bp.…”

“…In this regard, modular adaptor proteins such as PDZ (PSD-95/discs large/ZO-1) domain-containing proteins have drawn increasing attention due to their ability to form supramolecular complexes (7). We have previously shown that the regulatory interaction between CFTR and Na ϩ /H ϩ exchanger 3 (NHE3) through PDZ-based scaffolds is essential for the coordinated regulation of pancreatic bicarbonate secretion (8). In addition, it was found that a number of membrane transporters and receptors participating in pancreatic fluid formation, such as Na ϩ -HCO 3 Ϫ cotransporters (NBC), purinergic receptors, and secretin receptors have a PDZ-binding motif on their C terminus (9,10).…”

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

“…RNA (2 μg) was reverse-transcribed by using the One-step RT-PCR kit (Qiagen) with primers based on NHERF-1 and NHERF-2 mouse sequences. The primer sequences were as follows: NHERF-1 sense (5′-CTA AGC CAG GCC AGT TCA TCC GAG CAG T-3′) and antisense (5′-TGG GGT CAG AGG AGG AGG AGG AGG TAG A-3′), with a PCR product size of 447 bp (Ahn et al 2001); NHERF-2 sense (5′-CTCAATGGTGGCTCTGCGTGC-3′) and antisense (5′-TGATTTCTGGGCASTGGCAGG-3′), with a PCR product size of 346 bp (Yun et al 2002). The house-keeping gene, β-actin, was amplified as a control for the RT-PCR by using commercially available primers, giving a PCR product of 320 bp (Promega).…”