Biomedical subjects

M C Winter

Publications and source records attributed to M C Winter.

9 recordsLinked to original sources

PubMed1999

Histamine alters cadherin-mediated sites of endothelial adhesion.

We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.

Animals↗

PubMed1997

Stimulation of CFTR activity by its phosphorylated R domain.

Phosphorylation controls the activity of ion channels in many tissues. In epithelia, the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by phosphorylation of serine residues in its regulatory (R) domain and then gated by binding and hydrolysis of ATP by the nucleotide-binding domains. Current models propose that the unphosphorylated R domain serves as an inhibitory particle that occludes the pore, much like the inhibitory 'ball' in Shaker K+ channels; presumably, phosphorylation relieves this inhibition. Here we test this by adding an R-domain peptide to a CFTR variant in which much of the R domain had been deleted (CFTR-deltaR/S660A): in contrast to predictions, we found that adding an unphosphorylated R domain to CFTR-deltaR/S660A did not inhibit activity, whereas a phosphorylated R-domain peptide stimulated activity. To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity.

Adenosine Triphosphate↗

PubMed1995

Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels.

A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPi and three nonhydrolyzable PPi analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPi affected two distinct steps in channel regulation. First, PPi increased the rate at which channels opened. Second, once channels were open, PPi delayed their closure. PPi could only stimulate channels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPi or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.

Adenosine Triphosphate↗

PubMed1995

Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency.

Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments.

Adenosine Triphosphate↗

PubMed1994

Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.

3T3 Cells↗

PubMed1994

Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation.

Phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels require nucleoside triphosphates, such as ATP, to open. As the concentration of intracellular ATP increases, the probability of the channel being open (Po) increases. To better understand how ATP regulates the channel, we studied excised inside-out membrane patches that contained single, phosphorylated CFTR Cl- channels and examined the kinetics of gating at different concentrations of ATP. As the ATP concentration increased from 0.1 to 3 mM the mean closed time decreased, but mean open time did not change. Analysis of the data using histograms of open- and closed-state durations, the maximum likelihood method, and the log-likelihood ratio test suggested that channel behavior could be described by a model containing one open and two closed states (C1<==>C2<==>O). ATP regulated phosphorylated channels at the transition between the closed states C1 and C2: as the concentration of ATP increased, the rate of transition from C1 to C2 (C1-->C2) increased. In contrast, transitions from C2 to C1 and between C2 and the open state (O) were not significantly altered by ATP. Addition of ADP in the presence of ATP decreased the transition rate from C1 to C2 without affecting other transition rates. These data suggest that ATP regulates CFTR Cl- channels through an interaction that increases the rate of transition from the closed state to a bursting state in which the channel flickers back and forth between an open and a closed state (C2). This transition may reflect ATP binding or perhaps a step subsequent to binding.

3T3 Cells↗

PubMed1992

Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.

3T3 Cells↗

PubMed1992

Mastoparan activates apical chloride and potassium conductances, decreases cell volume, and increases permeability of cultured epithelial cell monolayers.

Mastoparan is a tetradecapeptide. Mastoparan added to the apical surface of monolayers of Madin-Darby canine kidney (MDCK) epithelial cells, cultured on micropore filters, activated ion transport and increased the permeability of the paracellular pathway across the monolayers. In monolayers of similar MDCK cells in which the basolateral membrane was permeabilized with Staphylococcus aureus alpha toxin (Staph. alpha toxin), the effects of mastoparan on apical membrane ion conductances were dependent on the presence of guanosine triphosphate (GTP). Mastoparan and GTP increased apical membrane chloride conductance more than potassium conductance, with very little change in sodium conductance. In intact monolayers, addition of barium to the apical bath prevented mastoparan activation of ion transport and the increase in paracellular permeability. Increasing bath potassium to 130 mM also reduced ion transport and prevented the increase in paracellular permeability. We hypothesized that these observations could be linked by mastoparan activation of apical chloride and potassium conductances, with consequent decreases in cell volume and resultant increases in paracellular permeability. Addition of 270 mM mannitol to isosmotic media to decrease cell volume decreased MDCK monolayer transepithelial resistance. Addition of mastoparan to monolayers of MDCK cells grown on micropore filters decreased cell volume to the same extent as addition of 270 mM mannitol to isosmotic media. Addition of the potassium channel inhibitor, barium, prevented the decrease in cell volume in response to mastoparan. Mastoparan activates apical membrane chloride and potassium conductances in MDCK cells. The loss of these ions from the cells decreases cell volume, and the decrease in cell volume increases the permeability of the paracellular pathway.

Animals↗

PubMed1991

Synergistic effects of a calcium ionophore and activators of protein kinase C on epithelial paracellular permeability.

Oxidants reversibly increase the paracellular permeability of Madin Darby canine kidney (MDCK) epithelial cell monolayers, and the decrease in resistance occurs within 10 to 15 min of initiating oxidant exposure. Oxidants also initiate hydrolysis of phosphatidylinositol in MDCK cells, with resultant increases in diacylglycerol and inositol phosphates. Phorbol esters and synthetic diacylglycerols increase the paracellular permeability of MDCK monolayers with a time course similar to the oxidants. In contrast, calcium ionophores increase MDCK monolayer paracellular permeability only after 2 to 3 h of exposure. Because the products of the oxidant-initiated phospholipid hydrolysis would be likely to both activate protein kinase C and increase cell calcium, we asked if ionomycin, a calcium ionophore, and phorbol esters or diacylglycerols, activators of protein kinase C, might not act in concert to alter MDCK monolayer paracellular permeability. When ionomycin was added alone to MDCK monolayers, there was an increase in cell calcium, activation of a lumen negative current, a limited transitory decrease in transepithelial resistance, but no increase in mannitol flux across the monolayers. When phorbol dibutyrate (PDBU) or oleyl acetyl glycerol (OAG) were added to MDCK monolayers, there was no current activated, there was a progressive decrease in transepithelial resistance, and there was an increase in mannitol flux across the monolayers which was evident within 20 to 40 min of adding the agent. When 1 microM ionomycin was added to the monolayers along with PDBU or OAG, there was a synergistic increase in paracellular permeability of the monolayers when compared to addition of ionomycin, PDBU, or OAG alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗