Biomedical subjects

Alice G M Bot

Publications and source records attributed to Alice G M Bot.

5 recordsLinked to original sources

PubMed2005

Activation of CFTR by ASBT-mediated bile salt absorption.

In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl- secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl- secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl- secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl- exit. TC-induced Cl- secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state.

Absorption↗

PubMed2004

Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel.

Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na(+) absorption mediated by Na(+)/H(+) exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na(+) absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g., cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current) across NHE3-null [NHE3(-)] murine small intestine and electroneutral Na(+) absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway, i.e., CFTR-null [CFTR(-)] or Na(+)-K(+)-2Cl(-) cotransporter-null [NKCC1(-)]. Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(-), compared with wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation was significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(-) and NKCC1(-) intestine, Na(+) absorption was significantly reduced compared with wild-type. NHE3 protein expression was decreased in the CFTR(-) intestine but was unchanged in the NKCC1(-) intestine, indicating that factors independent of expression also downregulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.

Animals↗

PubMed2004

Immunohistochemistry of CFTR in native tissues and primary epithelial cell cultures.

Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties. However, such data are of the highest importance to understand the pathophysiology of CF. The purpose of this article is to outline several assays for the characterization of primary epithelial cultures and to review different CFTR immunostaining protocols.

Cell Culture Techniques↗

PubMed2004

Biochemical methods to assess CFTR expression and membrane localization.

Detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein is usually a difficult task to accomplish due to the low levels of expression and high turnover that this membrane protein is submitted to in the cell. Common biochemical methods can be used for the detection of CFTR but several critical points must be taken into account. The scope of this article is to outline biochemical methods commonly used to assess CFTR expression, processing and membrane localization.

Biotinylation↗

PubMed2003

Autophosphorylation of cGMP-dependent protein kinase type II.

Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase (cGK) on multiple sites. Mass spectrometric based analyses, using a quadrupole time-of-flight-mass spectrometry instrument revealed that cGMP stimulated the in vitro phosphorylation of residues Ser110 and Ser114, and, at a slow rate, of Ser126 and Thr109 or Ser117, all located in the autoinhibitory region. In addition Ser445 was found to be phosphorylated in a cGMP-dependent manner, whereas Ser110 and Ser97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40-50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme.

Amino Acid Sequence↗