Involvement of cytoskeletal integrity in the regulation of Cl- and amylase secretion from rat parotid acinar cells.

The cytoskeleton serves as a signal modulator for Ca2+ and cAMP-regulated cell functions including the secretion of ions and granule contents. The interaction between Ca2+ and cAMP signaling systems potentiates amylase secretion and suppresses Cl- secretion in the parotid glands. In this study, we investigated the role of the cytoskeleton in the modulation of Cl- and amylase secretion from rat parotid acinar cells upon activation of each intracellular signaling system and their interaction. Cytochalasin D markedly inhibited the Ca2+-activated outwardly rectifying Cl- current at positive membrane potentials and carbachol (CCh)-induced Cl- currents in the whole-cell configuration at -80 mV, whereas colchicine enhanced Cl- currents. Cytochalasin D, but not colchicine, markedly inhibited CCh-induced Cl- secretion. Synergistic actions of CCh and forskolin on Cl- and amylase secretion were observed even in the presence of cytochalasin D. These results suggest that the synergistic effects of Ca2+ and cAMP signaling systems on amylase and Cl- secretion do not require actin filament integrity but that secretion by the two signals themselves does require actin filament integrity.

Molecular dissection of the butyrate action revealed the involvement of mitogen-activated protein kinase in cystic fibrosis transmembrane conductance regulator biogenesis.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which belongs to the superfamily of ATP-binding cassette transporters and uniquely possesses an additional large cytoplasmic domain [regulatory (R) domain]. CFTR inefficiently folds by means of co- and post-translational interactions with the cytosolic chaperones as well as luminal chaperones in the endoplasmic reticulum (ER). Aberrant folding and defective trafficking of the CFTR protein, which functions as an apical membrane Cl(-) channel, is the principal cause of cystic fibrosis. Recent data indicated that butyrate improves CFTR trafficking partly by regulating molecular chaperones; however, the precise mechanism of butyrate action remains elusive. In the present study, we examine the molecular aspect underlying the butyrate action in CFTR biogenesis by evaluating the expression and localization of the green fluorescent protein (GFP)-tagged CFTR transgenes in Cos7 cells. Our data show that butyrate significantly promoted stability of the ER-located form of GFP-wild-type (wt)-CFTR, followed by an increase in the amount of plasma membrane GFP-wt-CFTR. In contrast, the expression of the R domain deletion mutant GFP-DeltaR-CFTR was slightly increased by butyrate. The butyrate action on wt-CFTR expression was partially blocked by PD98059 (2'-amino-3'-methoxyflavone), a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), which is the upstream activator of extracellular-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). Furthermore, activation of ERK/MAPK by the coexpression of constitutively active MAPKK/MEK predominantly augmented the expression of wt-CFTR, but not of DeltaR-CFTR, induced by butyrate. These data suggest that butyrate may facilitate the biogenesis and trafficking of wt-CFTR by requiring the presence of the R domain and further involving active ERK/MAPK in its biogenesis.