Cyclic AMP and Chloride-dependent Regulation of the Apical Constitutive Secretory Pathway in Colonic Epithelial Cells

doi:10.1074/jbc.271.8.4381

Cyclic AMP and Chloride-dependent Regulation of the Apical Constitutive Secretory Pathway in Colonic Epithelial Cells (*)

From the Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

^§ To whom correspondence should be addressed:
Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL 35294
. Tel.: 205-934-3653; Fax: 205-934-1445.

Abstract

Epithelial cells of the colonic crypt engage in cAMP-mediated fluid and electrolyte secretion. In addition to participating in electrolyte transport, colonic crypt cells also synthesize and secrete a number of proteins and peptides that play a crucial role in mucosal homeostasis. In the present study we show that cAMP regulates not only electrolyte secretion but also polarized protein secretion in a tissue culture model of colonic crypt cells. We found that apical but not basolateral protein secretion was stimulated by a physiological activator of the cAMP pathway, vasoactive intestinal peptide, as well as by a cell-permeant analogue of cAMP (8-(4-chlorophenylthio)cAMP) at concentrations as low as 12.5 μM. Based on several criteria, we determined that the regulation of protein secretion by cAMP in HT29-CL19A cells occurs via stimulation of constitutive membrane traffic from the trans-Golgi network (TGN) to the apical cell surface. In addition, the regulation of apical protein secretion by cAMP was Cl-dependent with cAMP inhibiting rather than stimulating secretion in Cl-depleted cells. The locus of cAMP action on the secretory pathway is at least in part at the level of the TGN, where it stimulates the sialylation of α1-antitrypsin (i.e. one of the identified secretory proteins) in addition to the traffic of secretory proteins from the TGN to the apical cell surface. We propose that a cyclic AMP and Cl-dependent regulation of TGN acidification could modulate both sialylation and secretory vesicle budding at the TGN.

Footnotes

↵^¶ Established Investigator of the American Heart Association during the course of this study.
↵* This research was supported by Grant R464 PP#5 from the Cystic Fibrosis Foundation (to T. J.) and National Institutes of Health Grant DK50830 (to K. L. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ARF

ADP-ribosylation factor

VIP

vasoactive intestinal peptide

cpt-cAMP

8-(4-chlorophenylthio)-adenosine 3′:5′-cyclic monophosphate

TGN

trans-Golgi network

CFTR

cystic fibrosis transmembrane conductance regulator

AT

α1 antitrypsin

PAGE

polyacrylamide gel electrophoresis

IEF

isoelectric focusing

FBS

fetal bovine serum

TEMED

N,N,N',N'-tetramethylethylenediamine

DMEM

Dulbecco's modified Eagle's medium

MEBSS

modified Earle's buffered salt solution

BAPTA-AM

1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)-ester.
- Received October 12, 1995.
- Revision received December 11, 1995.