scholarly journals Discovery of Glycine Hydrazide Pore-occluding CFTR Inhibitors

2004 ◽  
Vol 124 (2) ◽  
pp. 125-137 ◽  
Author(s):  
Chatchai Muanprasat ◽  
N.D. Sonawane ◽  
Danieli Salinas ◽  
Alessandro Taddei ◽  
Luis J.V. Galietta ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Water Solubility ◽  
Fluid Secretion ◽  
Inhibition Mechanism ◽  
Inward Rectification ◽  
Loop Model ◽  
Inhibitory Potency ◽  
Voltage Dependent ◽  
Intestinal Fluid Secretion ◽  
Channel Open Time

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl− channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 μM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl− conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 μM at +60 mV to 5.6 μM at −60 mV. Apparent potency was reduced by lowering extracellular Cl− concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (−60 mV holding potential, 5 μM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5–8.0, where it exists predominantly as a monovalent anion with solubility ∼1 mM in water. Topical GlyH-101 (10 μM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 μg) reduced by ∼80% cholera toxin–induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTRinh-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.

Differences in jejunal and ileal response to E. coli enterotoxin: possible mechanisms

1989 ◽  
Vol 257 (1) ◽  
pp. G118-G123 ◽  
Author(s):  
M. B. Cohen ◽  
M. R. Thompson ◽  
R. A. Giannella
Keyword(s):  
High Performance ◽  
Time Course ◽  
Fluid Secretion ◽  
Close Correlation ◽  
Binding Activity ◽  
Secretory Response ◽  
Loop Model ◽  
E Coli ◽  
Heat Stable ◽  
Intestinal Fluid Secretion

Escherichia coli, which produce heat-stable enterotoxin (STa), cause intestinal fluid secretion as a mechanism of diarrhea. To determine the factors that modulate the intestinal secretory response, we first compared the time course of the STa-induced secretion in ligated in situ loops of rat jejunum and ileum. We found that the jejunal secretory response was brief (less than or equal to 30 min) while the ileal response to STa was sustained (greater than or equal to 3 h). We then compared the modification of purified STa that occurred in jejunum and ileum and found a close correlation between the continued presence of unmodified, authentic STa and continued fluid secretion in the ligated-loop model. At both sites alteration of STa was demonstrated by high-performance liquid chromatography profile and brush-border membrane binding activity. However, in the jejunum, the modification of STa was qualitatively different and quantitatively much greater. We conclude that the degree to which STa is inactivated or removed from the intestine correlates with the secretory response observed. Inactivation of STa may be a mechanism by which the host limits its secretory response.

scholarly journals The Capsule Encoding the viaB Locus Reduces Interleukin-17 Expression and Mucosal Innate Responses in the Bovine Intestinal Mucosa during Infection with Salmonella enterica Serotype Typhi

2007 ◽  
Vol 75 (9) ◽  
pp. 4342-4350 ◽  
Author(s):  
Manuela Raffatellu ◽  
Renato L. Santos ◽  
Daniela Chessa ◽  
R. Paul Wilson ◽  
Sebastian E. Winter ◽  
...  
Keyword(s):  
Mouse Model ◽  
Salmonella Enterica ◽  
Intestinal Inflammation ◽  
Fluid Secretion ◽  
Interleukin 17 ◽  
Loop Model ◽  
Wild Type ◽  
Ileal Loop ◽  
Chemokine Production

ABSTRACT The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica serotype Typhi. Wild-type serotype Typhi induces less CXC chemokine production in tissue culture models than does an isogenic viaB mutant. Here we investigated the in vivo relevance of these observations by determining whether the presence of the viaB region prevents inflammation in two animal models of gastroenteritis. Unlike S. enterica serotype Typhimurium, serotype Typhi or a serotype Typhi viaB mutant did not elicit marked inflammatory changes in the streptomycin-pretreated mouse model. In contrast, infection of bovine ligated ileal loops with a serotype Typhi viaB mutant resulted in more fluid accumulation and higher expression of the chemokine growth-related oncogene alpha (GROα) and interleukin-17 (IL-17) than did infection with the serotype Typhi wild type. There was a marked upregulation of IL-17 expression in both the bovine ligated ileal loop model and the streptomycin-pretreated mouse model, suggesting that this cytokine is an important component of the inflammatory response to infection with Salmonella serotypes. Introduction of the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GROα and IL-17 expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in reducing intestinal inflammation in vivo.

scholarly journals Distinct AMPA-Type Glutamatergic Synapses in Developing Rat CA1 Hippocampus

2010 ◽  
Vol 104 (4) ◽  
pp. 1899-1912 ◽  
Author(s):  
Elizabeth A. Stubblefield ◽  
Tim A. Benke
Keyword(s):  
Pyramidal Neurons ◽  
Age Groups ◽  
Inward Rectification ◽  
Decay Kinetics ◽  
Glutamatergic Synapses ◽  
Early Postnatal Development ◽  
Paired Pulse ◽  
Age Related ◽  
Hippocampal Ca1 ◽  
Voltage Dependent

We assessed synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) properties during synaptogenesis to describe the development of individual glutamatergic synapses on rat hippocampal CA1 principal neurons. Pharmacologically isolated AMPAR-mediated glutamatergic synaptic currents [evoked by stimulation of the Schaffer Collateral pathway, excitatory postsynaptic currents (EPSCs)], had significantly greater inward-rectification at ages P5–7 compared with P8–18. These inward rectifying EPSCs demonstrated paired-pulse dependent unblocking at positive holding potentials, consistent with voltage-dependent internal polyamine block. Measurements of paired-pulse facilitation did not support altered presynaptic properties associated with inward rectification. Using asynchronous EPSCs (aEPSCs) to analyze populations of individual synapses, we found that quantal amplitudes ( Q) increased across early postnatal development (P5-P18) and were directly modulated by increases in the number of activated receptors. Quantal AMPAR decay kinetics (aEPSC τdecays) exhibited the highest coefficient of variation (CV) from P5 to 7 and became markedly less variable at P8–18. At P5–7, faster quantal kinetics coexisted with much slower kinetics; only slower quantal kinetics were found at P8–18. This supports diverse quantal synaptic properties limited to P5–7. Multivariate cluster analysis of Q, CVτdecay, and median τdecay supported a segregation of neurons into two distinct age groups of P5–7 and P8–18, similar to the age-related segregation suggested by inward rectification. Taken together, these findings support synaptic, calcium permeable AMPARs at a subset of synapses onto CA1 pyramidal neurons exclusively at P5–7. These distinct synapses coexist with those sharing the properties of more mature synapses. These synapses disappear after P7 as activated receptor numbers increase with age.

Reduction of voltage-activated K+ currents by forskolin is not mediated via cAMP in pleural sensory neurons of Aplysia

1990 ◽  
Vol 64 (5) ◽  
pp. 1474-1483 ◽  
Author(s):  
D. A. Baxter ◽  
J. H. Byrne
Keyword(s):  
Adenylate Cyclase ◽  
Sensory Neurons ◽  
Water Solubility ◽  
Membrane Current ◽  
Peak Amplitude ◽  
Bath Application ◽  
Voltage Dependent ◽  
K Current ◽  
Derivatives Of ◽  
K Currents

1. Forskolin is often used to activate adenylate cyclase in studies relating adenosine 3',5'-cyclic monophosphate (cAMP) to the modulation of membrane current. There is growing concern, however, that some actions of forskolin are independent of cAMP. With the use of two-electrode voltage-clamp techniques, we compared the effects of analogues of cAMP to the effects of forskolin on K+ currents in somata of sensory neurons that were isolated from pleural ganglia of Aplysia californica. 2. Analogues of cAMP did not reduce the peak amplitude of either the transient K+ current (IA) or the voltage-dependent K+ current (IK.V). Analogues of cAMP did reduce the previously described cAMP-sensitive S K+ current (IK.S). In contrast, forskolin reduced the peak amplitude of both IA and IK.V. Furthermore, both IA and IK.V were reduced by 1,9-dideoxy-forskolin, a derivative of forskolin that does not activate adenylate cyclase. These results indicate that the effects of forskolin and 1,9-dideoxy-forskolin on IA and IK.V were not mediated via cAMP. 3. Bath application of a modified form of forskolin (7-deacetyl-6-[N-acetylglycyl]-forskolin), which has enhanced water solubility and activates adenylate cyclase, reduced IK.S, but did not alter either IA or IK.V. Thus it appears that certain derivatives of forskolin can be used to activate adenylate cyclase and avoid some of the nonspecific actions on membrane current that are associated with forskolin.

Sign in / Sign up

Export Citation Format

Share Document