cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is regulated by cAMP-dependent vesicle traffic and exocytosis to the apical membrane in some cell types, but this has not been demonstrated in the intestinal crypt. The distribution of CFTR, lactase (control), and fluid secretion were determined in rat jejunum after cAMP activation in the presence of nocodazole and primaquine to disrupt vesicle traffic. CFTR and lactase were localized by immunofluorescence, and surface proteins were detected by biotinylation of enterocytes. Immunoprecipitates from biotinylated and nonbiotinylated cells were analyzed by streptavidin detection and immunoblots. Immunolocalization confirmed a cAMP-dependent shift of CFTR but not lactase from a subapical compartment to the apical surface associated with fluid secretion that was reduced in the presence of primaquine and nocodazole. Analysis of immunoblots from immunoprecipitates after biotinylation revealed a 3.8 ± 1.7-fold ( P < 0.005) increase of surface-exposed CFTR after vasoactive intestinal peptide (VIP). These measurements provide independent corroboration supporting a role for vesicle traffic in regulating CFTR and cAMP-induced fluid transport in the intestine.

STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G

“…Because proteins critical to STa-elicited anion secretion are enriched in the villus epithelium of rat jejunum (NHE3, GCC, and cGKII) and CFTR is the only known substrate for cGKII (7,19,22,26), we hypothesized that STa and cGMP would regulate surface levels of CFTR in villus enterocytes by movement of CFTR from a subapical location to the cell surface similar to what we observed after cAMP stimulation in rat jejunum (3)(4)(5). Furthermore, while the role of PKA and cGKII phosphorylation in activating CFTR on the plasma membrane has been established, the physiological role of PKA or cGKII in regulating CFTR trafficking to the cell surface has not been established.…”

“…Cholera toxin and cAMP agonists activate PKA-dependent phosphorylation of CFTR (14). Cholera toxin and cAMP also stimulate the recruitment of CFTR from subapical vesicles to increase the number of CFTR transporters on the apical plasma membrane of both crypt and villus enterocytes, although the precise mechanisms responsible for CFTR recruitment from subapical vesicles to the plasma membrane remain unclear (3)(4)(5).…”

Identification of intestinal ion transport defects in microvillus inclusion disease

“…Under steady-state conditions in the intestine, CFTR is most abundant in subapical vesicles of crypt and villus enterocytes, with less expression on the BBM. In secretory diarrhea, second messengers stimulate exocytic insertion of CFTR from subapical vesicles to markedly increase its functional expression on the enterocyte BBM (4,6,18). A number of factors regulate CFTR function include apical recycling, cAMP, cGMP-regulated traffic, myosin motors including myosin VI (Myo6), myosin Ia (Myo1a), Rab GTPases, and STX3 (6,11,18,34,52,56).…”

VIP as a Corrector of CFTR Trafficking and Membrane Stability

“…Functional evidence of increased membrane CFTR density after PKC stimulation were also reported in the human colon cell line HT29 together with increased mucus secretion (Bajnath et al, 1995). Contrary to previous observations in intestinal epithelial cells (Ameen et al, 2003;Bradbury and Bridges, 1992), raising intracellular cAMP by forskolin had no effect on the amount of CFTR at the apical membrane of Calu-3 cells. It is thus evident that VIP effect on CFTR membrane insertion is coupled to different signaling pathways in airways and intestinal cells, with the latter having more complex regulation possibly depending on the cellular model considered.…”