369 research outputs found
Biochemical investigations on rat small intestinal epithelium : mitochondrial protein synthesis and antibiotics cyclic nucleotide regulation and function
Gastrointestinal disorders form an important category
of diseases with an often obscure etiology or biochemical
basis. Extension of the fundamental knowledge about structure
and function of the enterocyte is essential for a
better understanding of the intestinal pathophysiology and
may probably permit a more rational approach to prevention
or therapeutic treatment of these disease
Osmotic cell swelling-induced ATP release mediates the activation of extracellular signal-regulated protein kinase (Erk)-1/2 but not the activation of osmo-sensitive anion channels
Human intestine 407 cells respond to hypo-osmotic stress by the rapid
release of ATP into the extracellular medium. A difference in the time
course of activation as well as in the sensitivity to cytochalasin B
treatment and BAPTA-AM
[1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl
ester] loading suggests that ATP leaves the cell through a pathway
distinct from volume-regulated anion channels. To evaluate a putative role
for nucleotides as autocrinic/paracrinic factors in osmotic signalling,
the effects of extracellular ATP on the regulation of volume-sensitive
anion channels as well as on the hypotonicity-induced activation of
extracellular signal-regulated protein kinases (Erk-1/2) were
investigated. Micromolar concentrations of ATP were unable to elicit an
isotope efflux from (125)I(-)-loaded cells by itself, but strongly
potentiated the hypotonicity-provoked anion efflux through a
Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP
= ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of
P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly
inhibited both the osmotically induced isotope efflux and whole-cell Cl(-)
currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar
ATP but also by the purinoceptor antagonists suramin and reactive blue,
was observed most prominently at depolarizing holding potentials,
suggesting a direct interaction with volume-sensitive Cl(-) channels
rather than interaction with purinoceptors. Both ATP and UTP, at
submicromolar levels, were found to act as potent activators of Erk-1/2 in
intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath
greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not
affect the swelling-induced isotope efflux or whole-cell Cl(-) currents.
Furthermore, pre-treatment with suramin or reactive blue almost completely
prevented the hypo-osmotic activation of Erk-1/2. The results indicate
that extracellularly released ATP functions as an autocrinic/paracrinic
factor that mediates hypotonicity-induced Erk-1/2 activation but does not
serve as an activator of volume-sensitive compensatory Cl(-) currents
Animal models of cystic fibrosis
AbstractAnimal models of cystic fibrosis, in particular several different mutant mouse strains obtained by homologous recombination, have contributed considerably to our understanding of CF pathology. In this review, we describe and compare the main phenotypic features of these models. Recent and possible future developments in this field are discussed
N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II
The apical membrane of intestinal epithelial cells harbors a unique
isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key
regulator of ion transport systems, including the cystic fibrosis
transmembrane conductance regulator (CFTR)-chloride channel. To explore
the mechanism of cGK II membrane-anchoring, recombinant cGK II was
expressed stably in HEK 293 cells or transiently in COS-1 cells. In both
cell lines, cGK II was found predominantly in the particulate fraction.
Immunoprecipitation of solubilized cGK II did not reveal any other tightly
associated proteins, suggesting a membrane binding motif within cGK II
itself. The primary structure of cGK II is devoid of hydrophobic
transmembrane domains; cGK II does, however, contain a penultimate
glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling
showed that cGK II was indeed able to incorporate [3H]myristate. Moreover,
incubation of cGK II-expressing 293 cells with the myristoylation
inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the
proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%.
Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized
predominantly in the cytosol after transient expression in COS-1 cells.
The absence of the myristoyl group did not affect the specific enzyme
activity or the Ka for cGMP and only slightly enhanced the thermal
stability of cGK II. These results indicate that N-terminal myristoylation
fulfills a crucial role in directing cGK II to the membrane
Activation of ion transport by combined effects of ionomycin, forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes
The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca2+ pathways, (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl- conductance by protein kinase A and a basolateral K+ conductance by Ca2+. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K+ conductance. The synergism is caused by PKC-dependent increase of the apical Cl- conductance and Ca2+-dependent increase of the basolateral K+ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation, (d) Forskolin and P
Heat-stable enterotoxin receptor/guanylyl cyclase C is an oligomer consisting of functionally distinct subunits, which are non-covalently linked in the intestine
Guanylyl cyclase (GC) C is a heat-stable enterotoxin (STa) receptor with a
monomeric M(r) of approximately 140,000. We calculated from its
hydrodynamic parameters that an active GC-C complex has a M(r) of 393,000,
suggesting that GC-C is a trimer under native conditions. Both trimeric
and dimeric GC-C complexes were detected by 125I-STa binding and
SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The
GC activity and STa binding from intestinal brush border membranes
comigrated in gel filtration and velocity sedimentation with recombinant
GC-C. However, 125I-STa cross-linking demonstrated that STa receptors with
molecular masses of 52 and 74 kDa are non-covalently attached to GC in the
intestine. Radiation inactivation revealed different functional sizes for
basal GC activity, STa-stimulated GC activity, and STa binding (59,
210-240, and 32-52 kDa, respectively). At low radiation doses, basal GC
activity was stimulated, suggesting that GC-C is inhibited by a relatively
large, probably internal structure. These results suggest that STa may
activate GC-C by promoting monomer-monomer interaction (internal
"dimerization") within a homotrimeric GC-C complex, and that GC-C is
proteolytically modified in the brush border membrane but retains its
function
The C-terminus of the transmembrane MUC17 mucin binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine
The membrane bound mucins have a heavily O-glycosylated extracellular domain, a single pass membrane domain and a short cytoplasmic tail. Three of the membrane bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical for PDZ domain binding proteins. To identify PDZ proteins able to interact with the mucins, we screened PDZ domain arrays using YFP-tagged proteins. MUC17 exhibited a strong binding to PDZK1 whereas the binding to NHERF1 was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by mass spectrometry. This was mediated through the C-terminal PDZ-interaction site in MUC17 which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush border membrane staining in the wild-type mice compared to an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes
Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors
Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26Β°C), and 4 h or 6 h incubation at 37Β°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators
- β¦