cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR Activity

Chung Ming Tse, Jianyi Yin, Varsha Singh, Rafiquel Sarker, Ruxian Lin, Alan S. Verkman, Jerrold R. Turner, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

Background & Aims: SLC26A3 (DRA) is an electroneutral Cl - /HCO 3 - exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3′,5′-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO 3 - secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. Methods: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRA inh -A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl - /HCO 3 - exchanger and is inhibited by DRA inh -A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTR inh -172. Conclusions: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

Original languageEnglish (US)
Pages (from-to)641-653
Number of pages13
JournalCMGH
Volume7
Issue number3
DOIs
StatePublished - Jan 1 2019

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Cystic Fibrosis Transmembrane Conductance Regulator
Human Activities
Colon
Caco-2 Cells
HEK293 Cells
Colforsin
Adenosine
Colonic Neoplasms
Diarrhea
Cell Line
Antibodies
Proteins

Keywords

  • CFTR
  • Cl /HCO Exchange
  • Colon
  • Enteroids
  • Secretory Diarrhea

ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

Cite this

cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR Activity. / Tse, Chung Ming; Yin, Jianyi; Singh, Varsha; Sarker, Rafiquel; Lin, Ruxian; Verkman, Alan S.; Turner, Jerrold R.; Donowitz, Mark.

In: CMGH, Vol. 7, No. 3, 01.01.2019, p. 641-653.

Research output: Contribution to journalArticle

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abstract = "Background & Aims: SLC26A3 (DRA) is an electroneutral Cl - /HCO 3 - exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3′,5′-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO 3 - secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. Methods: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRA inh -A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl - /HCO 3 - exchanger and is inhibited by DRA inh -A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTR inh -172. Conclusions: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.",
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T1 - cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR Activity

AU - Tse, Chung Ming

AU - Yin, Jianyi

AU - Singh, Varsha

AU - Sarker, Rafiquel

AU - Lin, Ruxian

AU - Verkman, Alan S.

AU - Turner, Jerrold R.

AU - Donowitz, Mark

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Background & Aims: SLC26A3 (DRA) is an electroneutral Cl - /HCO 3 - exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3′,5′-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO 3 - secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. Methods: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRA inh -A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl - /HCO 3 - exchanger and is inhibited by DRA inh -A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTR inh -172. Conclusions: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

AB - Background & Aims: SLC26A3 (DRA) is an electroneutral Cl - /HCO 3 - exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3′,5′-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO 3 - secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. Methods: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRA inh -A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl - /HCO 3 - exchanger and is inhibited by DRA inh -A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTR inh -172. Conclusions: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

KW - CFTR

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KW - Colon

KW - Enteroids

KW - Secretory Diarrhea

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