Ben C. Tilly

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl− secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels was analyzed after expression of cGK II or cGK Iβ in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I− efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Iβ or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Iβ enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e.g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Iβ nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade

Hypo‐osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP‐kinase 2 activity and Hsp27 phosphorylation. Pre‐treatment of the cells with the p38 MAP kinase inhibitor SB‐203580 blocks this activation, indicating that the hypotonicity‐induced activation of MAPKAP kinase 2 is, similarly to that described for hyper‐osmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo‐osmotic treatment, the opening of compensatory Cl− channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo‐sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl− channel activation but instead may play a role in subsequent cellular repair processes.

Increased vesicle recycling in response to osmotic cell-swelling; cause and consequence of hypotonicity-provoked ATP release

Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1–43. In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2–3 min and lasting for ∼10–15 min. The hypotonicity-induced increase in membrane surface area, like the cell swelling-induced release of ATP (Van der Wijk, T., De Jonge, H. R., and Tilly, B. C. (1999) Biochem. J. 343, 579–586), was diminished after 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester loading or cytochalasin B treatment. Uptake of TRITC-dextrans, however, was not affected. Treatment of the cells with the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-specific protease Clostridium botulinum toxin F not only nearly eliminated the hypotonicity-induced increase in membrane surface area but also strongly diminished the release of ATP, indicating the involvement of regulated exocytosis. Both the ATP hydrolase apyrase and the MEK inhibitor PD098059 diminished the osmotic swelling-induced increase in membrane surface area as well as the subsequent uptake of TRITC-dextrans. Taken together, the results indicate that extracellular ATP is required for the hypotonicity-induced vesicle recycling and suggest that a positive feedback loop, involving purinergic activation of the Erk-1/2 pathway, may contribute to the release of ATP from hypo-osmotically stimulated cells.

Electrophysiological responses to bradykinin and microinjected inositol polyphosphates in neuroblastoma cells : Possible role of inositol 1,3,4-trisphosphate in altering membrane potential

Addition of bradykinin to mouse N1E‐115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several‐fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pre‐treatment of the cells with a Ca2+‐ionophore abolishes the hormone‐induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.

Signalling Mechanisms Involved in Volume Regulation of Intestinal Epithelial Cells

Most mammalian cells have developed compensatory mechanisms to respond to the variable osmotic stress caused by changes in the concentrations of intracellular osmo-active substances (e.g. glucose, amino acids, lactate) or by variations in the osmolarity of the surrounding medium. In response to osmotic cell swelling, the Regulatory Volume Decrease (RVD) is triggered and directs a reduction in the tonicity of the cell by the concerted opening of cation and anion selective ion channels. To date, the K+ and Cl- conductances activated upon hypo-osmotic stimulation have been characterised electrophysiologically in many different cell systems. The molecular identity of the channels however, as well as the mechanism(s) involved in their activation have not yet been fully clarified and may differ between cell types. In this review, we will evaluate the different signalling pathways activated by osmotic cell swelling and discuss their putative role(s) in ion channel regulation, in maintaining cellular volume homeostasis, and in auto- and paracrinic signal transduction, with emphasis on intestinal epithelial cells.

Expression and regulation of chloride channels in neonatal rat cardiomyocytes.

Using an 125I− efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Ca2+-sensitive Cl− conductance, triggered upon stimulation of the cells with endothelin-1 or Ca2+-ionophore; (2) a cAMP/protein kinase A-operated Cl− conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI− channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl− channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl− channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.

Cholesterol depletion and genistein as tools to promote F508delCFTR retention at the plasma membrane.

Background/aims: F508delCFTR-, but not wtCFTR-, expressing fibroblasts resemble Niemann Pick type C cells in the massive intracellular accumulation of free cholesterol. The recruitment and activation of F508delCFTR by cholesterol depletion was studied. Methods: Filipin staining, forskolin-stimulated anion efflux and FITC-dextran uptake were studied in control cells and fibroblasts treated with 2-hydroxypropyl β-cyclodextrin phosphatidylcholine large unilamellar vesicles to deplete cellular free cholesterol.Results: Treatment of F508delCFTR-, but not wtCFTR-, expressing fibroblasts with 2-hydroxypropyl β-cyclodextrin resulted in a reduction in cellular cholesterol and a potentiation of the forskolin-induced anion efflux. In addition, forskolin also promoted a massive increase in the rate of endocytosis in F508delCFTR fibroblasts, which was absent in genistein- or cyclodextrin-treated cultures.Conclusion: The results not only suggest that reducing cellular cholesterol may serve as pharmacotherapeutic tool in the treatment of cystic fibrosis but also reveal a novel mechanism for genistein regulation of F508delCFTR, i.e. retention by inhibition of endocytosis.

Chloride Transport in the Cystic Fibrosis Enterocyte

Molecular mechanisms of intestinal chloride channel regulation and potential abnormalities in electrogenic chloride secretion in intestinal epithelium from cystic fibrosis (CF) patients were investigated by a combination of Ussing chamber, vesicle transport and off-cell patch-clamp analysis. Short circuit current (Isc) measurements in normal and CF rectal biopsies provided evidence for i) a defect in the cAMP-provoked activation of chloride secretion and a (hyper)expression of cAMP-dependent potassium secretion in all CF patients examined (n = 11); ii) a defect in the carbachol-provoked chloride secretion and a (hyper)expression of carbachol-induced potassium secretion in 6/11 patients; iii) a residual (but still impaired) carbachol-induced chloride secretion in 5/11 CF patients (including 2 sibs). The latter class of CF patients appeared to consist genetically of compound heterozygotes for the major delta-F508 deletion, suggesting a correlation between the nature of the mutation in the CF gene and the severity of the chloride secretory defect in CF intestine. In our search for a regulatory function of GTP-binding (G-) proteins detected previously in the luminal membrane of rat and human intestinal epithelial cells, evidence was found for the presence of a GTP[S]-activatable- and GDP[S]-inhibitable chloride conductance in the apical membrane of rat enterocytes and human colonocytes. In excised patches of human colonocyt membranes, this G-proteine-sensitive chloride conductance was identified further as a novel type of chloride channel (20pS; inwardly rectifying) that was different from the 33pS outwardly rectifying chloride channel activatable by cAMP-dependent proteinkinase (PK-A) and voltage depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of Signal Transduction by Growth Factor Receptors

Immediate consequences of growth factor-receptor interaction include tyrosine-specific protein phosphorylations, inositol lipid breakdown and changes in cytoplasmic pH (pHi) and in the level of free Ca2+. The rise in pHi has a permissive effect on DNA synthesis and is mediated by Na+/H+ exchanger in the plasma membrane, which is turned on by protein kinase C. The Ca2+ signal is generated through the inositol lipid pathway and may contribute to the expression of certain proto-oncogenes. Monoclonal antibodies to the epidermal growth factor receptor can act as partial agonists in that they can induce tyrosine kinase activity without inducing inositol lipid breakdown and ionic changes. These antibodies fail to induce DNA synthesis, suggesting that tyrosine kinase activation is not sufficient for stimulation of cell proliferation.

Bradykinin-Induced Inositol Phosphate Metabolism in Human A431 Epidermoid Carcinoma Cells

Addition of the nonapeptide bradykinin to human A431 epidermoid carcinoma cells causes an immediate release of inositol phosphates and a rapid rise in [Ca2+]i. Half-maximal stimulation occurs at a bradykinin concentration of 4 nM. In the continuous presence of a saturating hormone concentration (1 μM) the inositol phosphate accumulation levels off within approximately 2 min; however, subsequent stimulation with histamine, another activator of phospholipase C, gives an additional increase in inositol phosphate production. Separation of the inositol phosphates by HPLC-anion exchange chromatography, reveals a rapid but transient accumulation of Ins(1,4,5)P3, followed by an increase in Ins (1,3,4,5)P4 and Ins(1,3,4)P3. Our data support a precursor/product-relationship between Ins(1,4,5)P3 and Ins(1,3,4)P3 with Ins(1,3,4,5)P4 as intermediate and argue against a messenger role of Ins(1,3,4,5)P4 and Ins(1,3,4)P3 in Ca2+ signalling.