Action of neltenexine on anion secretion in human airway epithelia

Neltenexine has been applied to human lung diseases such as chronic obstructive pulmonary disease (COPD) as a mucolytic agent. However, we have no information on the neltenexine action in bronchial epithelial cells. We studied the neltenexine action on the ion transport in human submucosal serous Calu-3 cells. Under a hyper-secreting condition caused by terbutaline (a beta2-adrenergic agonist), neltenexine diminished anion secretion by inhibiting the Cl- and HCO3- uptake via Na+/K+/2Cl- cotransporter and Na+/HCO3- cotransporter without blockade of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and also diminished anion secretion via stimulation of Cl-/HCO3- exchanger, which facilitates the extrusion of more CFTR-permeant anion, Cl-, with the uptake of less CFTR-permeant anion, HCO3-. Thus, neltenexine reduced the hyper-secretion to keep an appropriate fluid level in the airway, providing a possibility that neltenexine can be an effective drug in airway obstructive diseases by decreasing the airway resistance under a hyper-secreting condition.     

Phosphodiesterase 4 inhibitors modulate beta2-adrenoceptor agonist-induced human airway hyperresponsiveness

Chronic exposure of human isolated bronchi to beta2-adrenergic agonists, especially fenoterol, potentiates smooth muscle contraction in response to endothelin-1 (ET-1), a peptide implicated in chronic inflammatory airway diseases. 5'-Cyclic adenosine monophosphate (cAMP) pathways are involved in fenoterol-induced hyperresponsiveness. The present study investigated whether chronic elevation of intracellular cAMP by other pathways than beta2-adrenoceptor stimulation provokes bronchial hyperresponsiveness. Samples from eighteen human bronchi were sensitized to ET-1 by prolonged incubation with 0.1 microM fenoterol (15 h, 21 degrees C), or, under similar conditions, were incubated with a selective type-3 phosphodiesterase inhibitor (1 microM siguazodan), two selective type-4 phosphodiesterase inhibitors (0.1 microM rolipram and 0.1 microM cilomilast), a combination of fenoterol and rolipram (0.1 microM each) or of fenoterol and cilomilast (0.1 microM each). Rolipram and cilomilast, but not siguazodan, induced hyperresponsiveness (p < 0.01 and p < 0.05 vs. paired controls, respectively) similar to the fenoterol effect. Fenoterol-induced bronchial hyperresponsiveness was significantly enhanced by coincubation with cilomilast (p < 0.05 vs. fenoterol alone) but not with rolipram. Our results suggest that prolonged activation of intracellular cAMP through phosphodiesterase 4 inhibition induces hyperresponsiveness to ET-1 in human isolated bronchi. However, differences in subcellular localization of phosphodiesterase 4 may provoke divergent responsiveness patterns when human bronchi are continuously exposed to selective phosphodiesterase inhibitors with or without beta2-adrenergic agonists.     

Effects of endothelin-3 on intestinal ion transport

We investigated the effects of endothelin 3 (ET-3) on electrolyte transport in rat small intestine using a voltage clamp technique in Ussing’s chamber. ET-3 diminished potential difference (PD) and short circuit current (Isc). ET-3 did not affect PD or Isc in low Na⁺ and/or D-glucose-free medium. Phloridzine (an inhibitor of sodium-glucose cotransporter [SGLT1]) pretreatment abolished the effect of ET-3 on Isc. Methylene blue (a soluble guanylate cyclase inhibitor) or N-nitro-L-arginine methyl ester (a NOS inhibitor) pretreatment delayed the effect of ET-3 on PD and Isc. ET-3 enhanced NOS activity on enterocytes and systemic NO production. Then, ET-3 could inhibit SGLT1 with the participation of NO.     

Tryptamine-based human β3-adrenergic receptor agonists. Part 2: SAR of the methylene derivatives

A series of tryptamine derivatives with modified sulfonamide were designed, synthesized, and evaluated for their ability to stimulate cAMP accumulation in CHO cells expressing the cloned human β₃-adrenergic receptor (AR). For this series of compounds, our objective was to symmetrize the α-position of the tryptamine moiety maintaining its activity and reducing the cost of production. Compound 11h, having m-aminobenzene, exhibited excellent agonistic activity for β₃-AR with excellent subtype selectivity.     

Effect of somatostatin on electrogenic ion transport in the duodenum and colon of the mouse, Mus domesticus

In this study, we have used the mouse intestine and the Ussing short circuit technique to compare the effects and mechanism of action of somatostatin (SST, 0.1 μM) on cAMP- and Ca²⁺-mediated ion secretion in the duodenum and colon of the Swiss-Webster mouse. The cAMP-dependent secretagogues, prostaglandin E₂ (1 μM) and dibutyryl-cAMP (150 μM) increased short circuit current (I_sc) in both regions, but only the colonic response was inhibited by SST. This inhibition was independent of enteric nerves, suggesting a direct action on the epithelial cells. The Ca²⁺-dependent secretagogue carbachol (10 μM) stimulated a transient increase in I_sc in both intestinal segments. In the duodenum, SST partially inhibited this increase in I_sc and both the responses to carbachol and SST were independent of enteric nerves. In the colon, while SST inhibited the carbachol induced increase in I_sc, pre-treatment with tetrodotoxin (750 nM) profoundly inhibited the carbachol induced increase in I_sc, thus markedly reducing the inhibitory effect of SST. This indicates an involvement of the enteric nervous system in the response to carbachol and the action of SST in the colon. These data indicate marked regional differences within the mouse intestine of the effects of SST on ion secretion and demonstrate different mechanisms of action of SST in the duodenum and colon.     

The role of bestrophin in airway epithelial ion transport

The purpose of this study was to identify Cl- channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl- channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT-PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36Cl- flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl- channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.     

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.     

Dependency of the O2 consumption/O2 transport relationship on amino acid supply in sepsis

Summary In sepsis tissue O₂ uptake may be abnormally limited because of a depressed O₂ consumption/O₂ transport relationship. This study has been performed to assess patterns of O₂ consumption, CO₂ production and O₂ transport in septic patients undergoing total parenteral nutrition; more in particular, this study has investigated the interdependence between the patterns of blood O₂ uptake and simultaneous CO₂ release, and the availability of substrates (amino acids, glucose and fat). It has been shown that the O₂ consumption/O₂ transport relationship is significantly influenced by the exogenous amino acid load, which tends to increase O₂ uptake and O₂ consumption at any given O₂ transport, thus suggesting a favourable effect of amino acid administration on energy metabolism. The data on CO₂ production and CO₂ release, in addition to reconfirming the results of previous studies, have shown that the changes in O₂ uptake and in CO₂ production mediated by substrate doses have a quantifiable impact on blood O₂-CO₂ exchange interactions.     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

Phenotypic linkage between single-nucleotide polymorphisms of beta3-adrenergic receptor gene and NADH dehydrogenase subunit-2 gene,with special reference to eating behavior

The beta(3)-adrenergic receptor gene (BAR-3) allelic variant (Trp64Arg and Arg64Arg) is correlated with obesity or non-insulin-dependent diabetes mellitus. The mitochondrial NADH dehydrogenase subunit-2 gene (ND2) variant (Mt5178A) is associated with longevity or less susceptibility to adult-onset diseases. The frequencies of both the variants are high among the Japanese population. Cross-sectional analysis of these variants was conducted to determine if they correlated well with life-style-related phenotypes and nutrient intake. The body fat rate in the BAR-3 variant+ND2 variant group was higher than those rates in the BAR-3 normal+ND2 variant, BAR-3 normal+ND2 normal. The BAR-3 normal+ND2 variant group preferred much carbohydrate and less animal protein compared with other three groups. A combination of SNPs of the nuclear BAR-3 and the mitochondrial ND2 genes may affect eating behavior besides the biochemical and metabolic process of signal transduction and electron transfer system.     

Effects of Ag+ on ion transport by the corneal epithelium of the rabbit

Summary Exposure of thein vitro rabbit corneal epithelium to Ag⁺ by the addition of AgNO₃ (10^–7–10^–5)m) to the apical surface or by the use of imperfectly chlorided Ag/AgCl half-cells in Ussing-style membrane chambers, greatly increases short-circuit current and transepithelial potential. The early phase (the first 30 min) of the short-circuit current stimulation by Ag⁺ is linearly dependent on tear-side sodium concentration, is largely a result of a tenfold increase in net Na⁺ uptake and is incompletely inhibited by ouabain, suggesting that Ag⁺ increases cation (primarily Na⁺) conductance of the apical membrane. This mechanism for the Ag⁺ effect is supported by microelectrode experiments, wherein Ag⁺ depolarizes specifically the apical barrier potential and increases apical barrier conductance. A later phase in the effect (0.5–3 hr) is characterized by a gradual increase in³⁶Cl^– and¹⁴C-mannitol unidirectional fluxes, by a decline in epithelial resting potential and short-circuit current, by complete ouabain inhibition and by fit to saturation kinetics with respect to Na⁺ concentration in the bathing media. This pahse of the effect apparently reflects a nonselective opening of the paracellular pathway in the epithelium and is rate-limited by Na⁺ pump activity at the basolateral membrane. Both phases are associated with swelling of the corneal stroma and may be rapidly reversed using thiol agents (reduced glutathione and dithiothreitol). The results suggest that Ag⁺ may be useful in the study of cation transport by epithelia and the work provides basic physiological information that is pertinent to the prophylactic use of AgNO₃ in clinical ophthalmology.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Kinetics of ion transport in lipid membranes induced by lysine-valinomycin and derivatives

Summary Lysine-valinomycin and two N-acyl derivatives are compared with respect to their potency to transport Rb⁺ ions across thin lipid membranes. Lysine-valinomycin acts as a neutral ion carrier only above a pH of about 7 of the aqueous solutions, while at lower pH the molecules seem to be positively charged due to a protonation of the -NH₂ group of the lysine residue.A kinetic analysis based on voltage jump relaxation experiments and on the nonlinearity of the current-voltage characteristics showed that the conductance increment per carrier molecule for uncharged lysine-valinomycin is similar to that of natural valinomycin. The attachment of a rather bulky side group such as the dansyl or para-nitrobenzyloxycarbonyl group reduced by approximately one order of magnitude.Some of the relaxation data of the valinomycin analogues were influenced by an unspedfic relaxation of the pure lipid membrane. This structural relaxation represents a limitation to the possibility of analyzing specific transport systems in thin lipid membranes by the voltage jump or charge pulse techniques. It is shown that the time dependence of this structural relaxation — which was first published by Sargent (1975) — is at variance with a three capacitor equivalent circuit of the membrane, which was suggested by Coster and Smith (1974) on the basis of a.c. measurements. A modified equivalent circuit has been found to represent a satisfactory analogue for the current relaxation in the presence of valinomycin. It turned out, however, that such an equivalent circuit provides little insight into the molecular mechanism of transport.     

Adaptation of macrophages to exercise training improves innate immunity

The effects of 3-week exercise training on the functions of peritoneal macrophages from BALB/c mice were investigated. Lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and proinflammatory cytokine production in macrophages from trained mice was markedly higher than those from control mice. Meanwhile, exercise training decreased the steady state level of β₂-adrenergic receptor (β₂AR) mRNA in macrophages. Overexpression of β₂AR in the macrophage cell line RAW264 by transfecting with β₂AR cDNA suppressed NO synthase (NOS) II expression but dose not influenced proinflammatory cytokine expression. When expression of transfected β₂AR in RAWar cells was downregulated by a tetracycline repressor-regulated mammalian expression system, NOS II mRNA expression was significantly increased; this suggested that the changes in the β₂AR expression level in macrophages associated with exercise training play a role in the regulation of NO production following LPS stimulation. These findings indicate that exercise training improves macrophage innate immune function in a β₂AR-dependent and -independent manner.     

Anoctamin 1 in secretory epithelia

Fluid and electrolyte releasing from secretory epithelia are elaborately regulated by orchestrated activity of ion channels. The activity of chloride channel at the apical membrane decides on the direction and the rate of secretory fluid and electrolyte. Chloride-dependent secretion is conventionally associated with intracellular increases in two second messengers, cAMP and Ca²⁺, responding to luminal purinergic and basolateral adrenergic or cholinergic stimulation. While it is broadly regarded that cAMP-dependent Cl⁻ secretion is regulated by cystic fibrosis transmembrane conductance regulator (CFTR), Ca²⁺-activated Cl⁻ channel (CaCC) had been veiled for quite some time. Now, Anoctamin 1 (ANO1 or TMEM16A) confers Ca²⁺-activated Cl⁻ currents. Ano 1 and its paralogs have been actively investigated for multiple functions underlying Ca²⁺-activated Cl⁻ efflux and fluid secretion in a variety of secretory epithelial cells. In this review, we will discuss recent advances in the secretory function and signaling of ANO1 in the secretory epithelia, such as airways, intestines, and salivary glands.