Antidiarrheal Efficacy and Cellular Mechanisms of a Thai Herbal Remedy

Screening of herbal remedies for Cl⁻ channel inhibition identified Krisanaklan, a herbal extract used in Thailand for treatment of diarrhea, as an effective antidiarrheal in mouse models of secretory diarrheas with inhibition activity against three Cl⁻ channel targets. Krisanaklan fully inhibited cholera toxin-induced intestinal fluid secretion in a closed-loop mouse model with ∼50% inhibition at a 1∶50 dilution of the extract. Orally administered Krisanaklan (5 µL/g) prevented rotavirus-induced diarrhea in neonatal mice. Short-circuit current measurements showed full inhibition of cAMP and Ca²⁺ agonist-induced Cl⁻ conductance in human colonic epithelial T84 cells, with ∼50% inhibition at a 1∶5,000 dilution of the extract. Krisanaklan also strongly inhibited intestinal smooth muscle contraction in an ex vivo preparation. Together with measurements using specific inhibitors, we conclude that the antidiarrheal actions of Krisanaklan include inhibition of luminal CFTR and Ca²⁺-activated Cl⁻ channels in enterocytes. HPLC fractionation indicated that the three Cl⁻ inhibition actions of Krisanaklan are produced by different components in the herbal extract. Testing of individual herbs comprising Krisanaklan indicated that agarwood and clove extracts as primarily responsible for Cl⁻ channel inhibition. The low cost, broad antidiarrheal efficacy, and defined cellular mechanisms of Krisanaklan suggests its potential application for antisecretory therapy of cholera and other enterotoxin-mediated secretory diarrheas in developing countries.     

Hydroxyxanthone as an inhibitor of cAMP-activated apical chloride channel in human intestinal epithelial cell

AimsPrevious investigation showed that polyphenols abundantly found in many plants could inhibit Cl^? secretion. The present study was aimed to investigate the effect of phenol containing xanthone derivatives on cAMP-activated intestinal Cl^? secretion and evaluate potential benefits of these compounds in the treatment of cholera.Main methodsFour hydroxy xanthones were synthesized via oxidative coupling reaction of the corresponding ortho-hydroxybenzoic acids and hydroxyphenols. Short-circuit current and apical Cl^? current measurements across monolayers of human intestinal epithelial (T84) cell and Fisher rat thyroid cells transfected with human CFTR (FRT-hCFTR cell) were performed to determine the effect of hydroxyxanthones on cAMP-activated Cl^? secretion. Intracellular cAMP was measured by immunoassay methods. Anti-diarrheal efficacy was evaluated using closed loop model of cholera.Key findingsAmong the tested xanthones, 1,3,6-trihydroxyxanthone (THX-001) was found to be the most potent derivative in the inhibition of cAMP-activated Cl^? secretion across T84 cell monolayers (IC₅₀ ~ 100 μM). Electrophysiological analysis of T84 cells and FRT-hCFTR cells revealed that THX-001 targeted two distinct cAMP-activated Cl^? channels in the apical membrane of T84 cells, namely, CFTR and inward rectifying Cl^? channel (IRC). In contrast, THX-001 had no effect on intracellular cAMP levels in these cells. Importantly, THX-001 completely abolished cholera toxin-induced Cl^? secretion across T84 cell monolayers and significantly inhibited cholera toxin-induced intestinal fluid secretion in mouse closed loop models.SignificanceThis study revealed that hydroxyxanthone represents another chemical class of polyphenolic compounds that may hold promise as anti-secretory therapy for cholera.     

The Epac1 Signaling Pathway Regulates Cl? Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

The apical membrane of intestinal epithelia expresses intermediate conductance K⁺ channel (KCNN4), which provides the driving force for Cl⁻ secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl⁻ secretion via stimulation of Rap2-phospholipase Cϵ-[Ca²⁺]_i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl⁻ secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl⁻ secretion and apical K⁺ conductance (I_K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2′-O- methyladenosine 3′,5′-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K⁺ channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I_K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I_K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.     

Why Mouse Airway Submucosal Gland Serous Cells Do Not Secrete Fluid in Response to cAMP Stimulation

Airway submucosal glands are important sites of cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl⁻) channel expression and fluid secretion in the airway. Whereas both mouse and human submucosal glands and their serous acinar cells express CFTR, human glands and serous cells secrete much more robustly than mouse cells/glands in response to cAMP-generating agonists such as forskolin and vasoactive intestinal peptide. In this study, we examined mouse and human serous acinar cells to explain this difference and reveal further insights into the mechanisms of serous cell secretion. We found that mouse serous cells possess a robust cAMP-activated CFTR-dependent Cl⁻ permeability, but they lack cAMP-activated calcium (Ca²⁺) signaling observed in human cells. Similar to human cells, basal K⁺ conductance is extremely small in mouse acinar cells. Lack of cAMP-activated Ca²⁺ signaling in mouse cells results in the absence of K⁺ conductances required for secretion. However, cAMP activates CFTR-dependent fluid secretion during low-level cholinergic stimulation that fails to activate secretion on its own. Robust CFTR-dependent fluid secretion was also observed when cAMP stimulation was combined with direct pharmacological activation of epithelial K⁺ channels with 1-ethyl-2-benzimidazolinone (EBIO). Our data suggest that mouse serous cells lack cAMP-mediated Ca²⁺ signaling to activate basolateral membrane K⁺ conductance, resulting in weak cAMP-driven serous cell fluid secretion, providing the likely explanation for reduced cAMP-driven secretion observed in mouse compared with human glands.     

Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

Intestinal Cl⁻ secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca²⁺]_i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl⁻ secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I_sc) measurement in response to agonist-stimulated Cl⁻ secretion. FSK-stimulated Cl⁻ secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca²⁺]_i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca²⁺]_i, activated Ras-related protein 2, and induced Cl⁻ secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I_sc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl⁻ secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl⁻ conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl⁻>Br⁻>I⁻ permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca²⁺]_i signaling pathway is involved in cAMP-stimulated Cl⁻ secretion, which is carried by a novel, previously undescribed Cl⁻ channel.     

Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon

Excessive Cl⁻ secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl⁻ secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl⁻ secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl⁻ secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K⁺ channel and apical Cl⁻ channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC₅₀ values of approximately 1 nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl⁻ currents were unaffected by 17β-estradiol treatment while basolateral K⁺ current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl⁻ secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl⁻ secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.     

Chloroquine Stimulates Cl? Secretion by Ca2+ Activated Cl? Channels in Rat Ileum

Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current (I_SC) in rat ileum at lower concentration (≤5×10⁻⁴ M ) but induced a decrease at higher concentrations (≥10⁻³ M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in I_SC was partly reduced by amiloride(10⁻⁴ M), a blocker of epithelial Na⁺ channels. Furosemide (10⁻⁴ M), an inhibitor of Na⁺-K⁺ -2Cl⁻ co-transporter, also inhibited the increased I_SC response to CQ, whereas another Cl⁻ channel inhibitor, CFTR(inh)-172(10⁻⁵M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10⁻⁴M), a relatively specific Ca²⁺-activated Cl⁻ channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in I_SC were also abolished by thapsigargin(10⁻⁶M), a Ca²⁺ pump inhibitor and in the absence of either Cl⁻ or Ca²⁺ from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca²⁺ in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl⁻ secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

Intracellular Cl⁻ concentrations ([Cl⁻]_i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl⁻ is accumulated by the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1), resulting in a [Cl⁻]_i above electrochemical equilibrium and a depolarizing Cl⁻ efflux upon Cl⁻ channel opening. Here, we investigate the [Cl⁻]_i and function of Cl⁻ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1^−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl⁻]_i of WT TG neurons indicated active NKCC1-dependent Cl⁻ accumulation. Gamma-aminobutyric acid (GABA)_A receptor activation induced a reduction of [Cl⁻]_i as well as Ca²⁺ transients in a corresponding fraction of TG neurons. Ca²⁺ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca²⁺ channels (VGCCs). Ca²⁺ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1^−/− TG neurons, but elevated under conditions of a lowered [Cl⁻]_o suggesting a Cl⁻-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca²⁺-activated Cl⁻ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca²⁺ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1^−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca²⁺-activated Cl⁻-dependent signal amplification mechanism in TG neurons that requires intracellular Cl⁻ accumulation by NKCC1 and the activation of CaCCs.     

Activated PKCδ and PKC? Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl? Cotransporter NKCC1

The basolateral Na⁺-K⁺-2Cl⁻ cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ϵ) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCϵ- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCϵ-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCϵ, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.     

Disruption of the K+ Channel β-Subunit KCNE3 Reveals an Important Role in Intestinal and Tracheal Cl? Transport

The KCNE3 β-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K⁺ channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K⁺ channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K⁺ channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3^−/− mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl⁻ secretion through basolateral recycling of K⁺ ions and by increasing the electrochemical driving force for apical Cl⁻ exit. Indeed, cAMP-stimulated electrogenic Cl⁻ secretion across tracheal and intestinal epithelia was drastically reduced in kcne3^−/− mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3^−/− mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.     

Cl? and K+ channels and their role in primary brain tumour biology

Profound cell volume changes occur in primary brain tumours as they proliferate, invade surrounding tissue or undergo apoptosis. These volume changes are regulated by the flux of Cl⁻ and K⁺ ions and concomitant movement of water across the membrane, making ion channels pivotal to tumour biology. We discuss which specific Cl⁻ and K⁺ channels are involved in defined aspects of glioma biology and how these channels are regulated. Cl⁻ is accumulated to unusually high concentrations in gliomas by the activity of the NKCC1 transporter and serves as an osmolyte and energetic driving force for volume changes. Cell volume condensation is required as cells enter M phase of the cell cycle and this pre-mitotic condensation is caused by channel-mediated ion efflux. Similarly, Cl⁻ and K⁺ channels dynamically regulate volume in invading glioma cells allowing them to adjust to small extracellular brain spaces. Finally, cell condensation is a hallmark of apoptosis and requires the concerted activation of Cl⁻ and Ca²⁺-activated K⁺ channels. Given the frequency of mutation and high importance of ion channels in tumour biology, the opportunity exists to target them for treatment.     

Cell Swelling Activates the K+ Conductance and Inhibits the Cl? Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium

Necturus gallbladder epithelial cells bathed in 10 mM HCO₃/1% CO₂ display sizable basolateral membrane conductances for Cl⁻ (G_Cl ^b) and K ⁺ (G_K ^b). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K ⁺/Cl⁻ selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca²⁺]_i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca²⁺ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (V_cs) produced a significant increase in the change in V_cs elicited by lowering basolateral solution [Cl⁻], whereas hyperpolarization of V_cs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases G_K ^b and decreases G _Cl ^b. These two effects appear to be linked, i.e., the increase in G _K ^b produces membrane hyperpolarization, which in turn reduces G _Cl ^b. ( b) Hyperosmotic shrinkage has the opposite effects on G_K ^b and G _Cl ^b. ( c) Cell swelling causes a transient increase in [Ca²⁺]_i, but this response may not be necessary for the increase in G_K ^b during cell swelling.     

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H₂S) donor Na₂S with IC₅₀ values of ∼2.4·10⁻⁵ and ∼7.9·10⁻⁵ m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H₂S does not interfere with substrate or cofactor binding. The IC₅₀ decreased to ∼1.5·10⁻⁵ m at pH 6.0 and increased to ∼8.3·10⁻⁵ m at pH 8.0. Preincubation of concentrated nNOS with H₂S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H₂S and NO. Likewise, NADPH oxidation was inhibited with an IC₅₀ of ∼1.9·10⁻⁵ m in the presence of Arg and BH4 but exhibited much higher IC₅₀ values (∼1.0–6.1·10⁻⁴ m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na₂S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC₅₀ ∼ 1.3–2.0·10⁻⁵ m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H₂S/NO at modest concentrations of H₂S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H₂S formation.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Molecular Mechanisms of Calcium-sensing Receptor-mediated Calcium Signaling in the Modulation of Epithelial Ion Transport and Bicarbonate Secretion

Epithelial ion transport is mainly under the control of intracellular cAMP and Ca²⁺ signaling. Although the molecular mechanisms of cAMP-induced epithelial ion secretion are well defined, those induced by Ca²⁺ signaling remain poorly understood. Because calcium-sensing receptor (CaSR) activation results in an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) but a decrease in cAMP levels, it is a suitable receptor for elucidating the mechanisms of [Ca²⁺]_cyt-mediated epithelial ion transport and duodenal bicarbonate secretion (DBS). CaSR proteins have been detected in mouse duodenal mucosae and human intestinal epithelial cells. Spermine and Gd³⁺, two CaSR activators, markedly stimulated DBS without altering duodenal short circuit currents in wild-type mice but did not affect DBS and duodenal short circuit currents in cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice. Clotrimazole, a selective blocker of intermediate conductance Ca²⁺-activated K⁺ channels but not chromanol 293B, a selective blocker of cAMP-activated K⁺ channels (KCNQ1), significantly inhibited CaSR activator-induced DBS, which was similar in wild-type and KCNQ1 knockout mice. HCO₃⁻ fluxes across epithelial cells were activated by a CFTR activator, but blocked by a CFTR inhibitor. CaSR activators induced HCO₃⁻ fluxes, which were inhibited by a receptor-operated channel (ROC) blocker. Moreover, CaSR activators dose-dependently raised cellular [Ca²⁺]_cyt, which was abolished in Ca²⁺-free solutions and inhibited markedly by selective CaSR antagonist calhex 231, and ROC blocker in both animal and human intestinal epithelial cells. Taken together, CaSR activation triggers Ca²⁺-dependent DBS, likely through the ROC, intermediate conductance Ca²⁺-activated K⁺ channels, and CFTR channels. This study not only reveals that [Ca²⁺]_cyt signaling is critical to modulate DBS but also provides novel insights into the molecular mechanisms of CaSR-mediated Ca²⁺-induced DBS.     

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca²⁺ concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca²⁺ in isolated mouse vomeronasal sensory neurons in the absence of intracellular K⁺. In whole-cell recordings, the average current in 1.5 µM Ca²⁺ and symmetrical Cl⁻ was −382 pA at −100 mV. Ion substitution experiments and partial blockade by commonly used Cl⁻ channel blockers indicated that Ca²⁺ activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca²⁺-activated Cl⁻ channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca²⁺-activated Cl⁻ channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre–loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca²⁺-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca²⁺-activated Cl⁻ currents in mouse vomeronasal sensory neurons.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Intracellular Cl? Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl⁻. The purpose of the present study was to determine (a) whether Cl⁻ plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl⁻]_i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl⁻- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na⁺ and 0–194 mM Cl⁻. The artificial seawater bathing the BMF initially contained no Na⁺. After dialysis was halted, adding 50 mM Na⁺ to the artificial seawater caused an amiloride-sensitive pH_i increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J _Na-H) increased with increasing [Cl⁻]_i under both normo- and hypertonic conditions, with similar apparent K _m values (∼120 mM). However, the maximal J _Na-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH_i requires Cl⁻ under both normo- and hypertonic conditions, but at any given [Cl⁻]_i, J _Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl⁻]_i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl⁻-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl⁻-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl⁻, dialyzing with GTPγS or AlF₃, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl⁻-depleted fibers, the Cl⁻-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF₃ indicates that the G protein is a heterotrimeric G protein.