Phosphatidylinositol 4,5-bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus oocytes.

KEY POINTS: We previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 ) directly stimulates heterologously expressed electrogenic Na(+)/bicarbonate cotransporter NBCe1-A in an excised macropatch from the Xenopus oocyte, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate/Ca(2+). In the current study, we expand on a previous observation that PIP2 may also directly stimulate NBCe1 in the intact oocyte. In this study on oocytes, we co-expressed either NBCe1-B or -C and a voltage-sensitive phosphatase (VSP), which depletes PIP2 without changing inositol 1,4,5-trisphosphate, and monitored NBCe1-mediated currents with the two-electrode voltage-clamp technique or pHi changes using Vm/pH-sensitive microelectrodes. Activating VSP inhibited NBCe1-B and -C outward currents and NBCe1-mediated pHi increases, and changes in NBCe1 activity paralleled changes in surface PIP2. This study is a quantitative assessment of PIP2 itself as a regulator of NBCe1-B and -C in the intact cell, and represents the first use of VSP to characterize the PIP2 sensitivity of a transporter. These data combined with our previous work demonstrate that NBCe1-B and -C are regulated by two PIP2-mediated signalling pathways. Specifically, a decrease in PIP2 per se can inhibit NBCe1, whereas hydrolysis of PIP2 to inositol 1,4,5-trisphosphate/Ca(2+) can stimulate the transporter. ABSTRACT: The electrogenic Na(+)/bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pHi) and extracellular pH (pHo), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) directly stimulates NBCe1-A in an excised macropatch, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate (InsP3)/Ca(2+). In the current study, we used the two-electrode voltage-clamp technique alone or in combination with pH/voltage-sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP2 to characterize the PIP2 sensitivity of NBCe1-B and -C in whole oocytes by co-expressing a voltage-sensitive phosphatase (VSP) that decreases PIP2 and bypasses the InsP3/Ca(2+) pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1-B/C current, consistent with an initial rapid depolarization-induced NBCe1 activation, and then a subsequent slower VSP-mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP2 resynthesis as measured with a PIP2-sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca(2+) did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP3/Ca(2+) were not responsible. This study demonstrates for the first time that depleting PIP2 per se inhibits NBCe1 activity. The data in conjunction with previous findings implicate a dual PIP2 regulatory pathway for NBCe1 involving both PIP2 itself and generated InsP3/Ca(2+).

ATP-independent CFTR channel gating and allosteric modulation by phosphorylation.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.

Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease.

Pathophysiological anomalies in autosomal dominant and recessive forms of polycystic kidney disease (PKD) may derive from impaired function/formation of the apical central monocilium of ductal epithelia such as that seen in the Oak Ridge polycystic kidney or orpk (Ift88(Tg737Rpw)) mouse and its immortalized cell models for the renal collecting duct. According to a previous study, Na/H exchanger (NHE) activity may contribute to hyperabsorptive Na(+) movement in cilium-deficient ("mutant") cortical collecting duct principal cell monolayers derived from the orpk mice compared with cilium-competent ("rescued") monolayers. To examine NHE activity, we measured intracellular pH (pH(i)) by fluorescence imaging with the pH-sensitive dye BCECF, and used a custom-designed perfusion chamber to control the apical and basolateral solutions independently. Both mutant and rescued monolayers exhibited basolateral Na(+)-dependent acid-base transporter activity in the nominal absence of CO(2)/HCO(3)(-). However, only the mutant cells displayed appreciable apical Na(+)-induced pH(i) recoveries from NH(4)(+) prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pH(i) dependence of basolateral cariporide/HOE-694-sensitive NHE activity under our experimental conditions was similar in both mutant and rescued cells, and 3.5- to 4.5-fold greater than apical HOE-sensitive NHE activity in the mutant cells (pH(i) 6.23-6.68). Increased apical NHE activity correlated with increased apical NHE1 expression in the mutant cells, and increased apical localization in collecting ducts of kidney sections from orpk vs. control mice. A kidney-specific conditional cilium-knockout mouse produced a more acidic urine compared with wild-type littermates and became alkalotic by 28 days of age. This study provides the first description of altered NHE activity, and an associated acid-base anomaly in any form of PKD.

PIP2 hydrolysis stimulates the electrogenic Na+-bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus laevis oocytes.

Electrogenic Na(+)-bicarbonate cotransporter NBCe1 variants contribute to pH(i) regulation, and promote ion reabsorption or secretion by many epithelia. Most Na(+)-coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from Xenopus laevis oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH- and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP(2) on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP(2) (10 µM estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO(3)(-)-induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP(2) hydrolysis and endoplasmic reticulum (ER) Ca(2+) release. Stimulation by PIP(2) injection was mimicked by injecting IP(3), but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca(2+) with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca(2+) channels (SOCCs) to trigger a Ca(2+) influx mimicked the PIP(2)/IP(3) stimulation of the B and C variants. Activating the endogenous G(q) protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca(2+)-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH(i) studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH(i)-recovery rate from a CO(2)-induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP(3)-induced stimulation of NBCe1-C. In summary, injecting PIP(2) stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP(3)/Ca(2+) that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP(2) regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP(2) on at least NBCe1-A, as well as an indirect stimulatory effect of IP(3)/Ca(2+) on the B and C variants.

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates the electrogenic Na/HCO3 cotransporter NBCe1-A expressed in Xenopus oocytes.

Bicarbonate transporters are regulated by signaling molecules/ions such as protein kinases, ATP, and Ca(2+). While phospholipids such as PIP(2) can stimulate Na-H exchanger activity, little is known about phospholipid regulation of bicarbonate transporters. We used the patch-clamp technique to study the function and regulation of heterologously expressed rat NBCe1-A in excised macropatches from Xenopus laevis oocytes. Exposing the cytosolic side of inside-out macropatches to a 5% CO(2)/33 mM HCO(3)(-) solution elicited a mean inward current of 14 pA in 74% of macropatches attached to pipettes (-V(p) = -60 mV) containing a low-Na(+), nominally HCO(3)(-)-free solution. The current was 80-90% smaller in the absence of Na(+), approximately 75% smaller in the presence of 200 microM DIDS, and absent in macropatches from H(2)O-injected oocytes. NBCe1-A currents exhibited time-dependent rundown that was inhibited by removing Mg(2+) in the presence or absence of vanadate and F(-) to reduce general phosphatase activity. Applying 5 or 10 microM PIP(2) (diC8) in the presence of HCO(3)(-) induced an inward current in 54% of macropatches from NBC-expressing, but not H(2)O-injected oocytes. PIP(2)-induced currents were HCO(3)(-)-dependent and somewhat larger following more NBCe1-A rundown, 62% smaller in the absence of Na(+), and 90% smaller in the presence of 200 microM DIDS. The polycation neomycin (250-500 microM) reduced the PIP(2)-induced inward current by 69%; spermine (100 microM) reduced the current by 97%. Spermine, poly-D-lysine, and neomycin all reduced the baseline HCO(3)(-)-induced inward currents by as much as 85%. In summary, PIP(2) stimulates NBCe1-A activity, and phosphoinositides are regulators of bicarbonate transporters.

Sodium-bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport.

In this study, we examined the effect of bicarbonate transporters on ammonium/ammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mm ouabain and 0.2 mM bumetanide to minimize carrier-mediated NH(4)(+) transport, and the intracellular accumulation of (14)C-methylammonium/methylammonia ((14)C-MA) was determined. In CO(2)/HCO(3)(-)-free solution, cells at normal pH briefly accumulated (14)C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)(-) solution, however, cells markedly accumulated (14)C-MA over the experimental period of 30 min. This CO(2)/HCO(3)(-)-dependent accumulation was reduced by the bicarbonate transporter blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS; 0.5 mM). Replacing Cl(-) with gluconate reduced the accumulation, but the reduction was more substantial in the presence of DIDS. Incubation of cells at pH 6.8 (adjusted with NaHCO(3) in 5% CO(2)) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for control cells. The presence of DIDS reduced (14)C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH(4)Cl-containing media. The Cl(-)-HCO(3)(-) exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated (14)C-MA transport, which was abolished by replacing Na(+). Two-electrode voltage clamp of oocytes exhibited negligible current after NH(4)Cl application. These results suggest that DIDS-sensitive HCO(3)(-) extrusion normally governs NH(4)(+)/NH(3) uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO(3)(-) extrusion is inactivated, while NBCn1 is upregulated to stimulate NH(4)(+) transport.

Reduction of AMPA receptor activity on mature oligodendrocytes attenuates loss of myelinated axons in autoimmune neuroinflammation.

Glutamate dysregulation occurs in multiple sclerosis (MS), but whether excitotoxic mechanisms in mature oligodendrocytes contribute to demyelination and axonal injury is unexplored. Although current treatments modulate the immune system, long-term disability ensues, highlighting the need for neuroprotection. Glutamate is elevated before T2-visible white matter lesions appear in MS. We previously reported that myelin-reactive T cells provoke microglia to release glutamate from the system xc - transporter promoting myelin degradation in experimental autoimmune encephalomyelitis (EAE). Here, we explore the target for glutamate in mature oligodendrocytes. Most glutamate-stimulated calcium influx into oligodendrocyte somas is AMPA receptor (AMPAR)-mediated, and genetic deletion of AMPAR subunit GluA4 decreased intracellular calcium responses. Inducible deletion of GluA4 on mature oligodendrocytes attenuated EAE and loss of myelinated axons was selectively reduced compared to unmyelinated axons. These data link AMPAR signaling in mature oligodendrocytes to the pathophysiology of myelinated axons, demonstrating glutamate regulation as a potential neuroprotective strategy in MS.

Effects of acute hypoxia on intracellular-pH regulation in astrocytes cultured from rat hippocampus.

We used the pH-sensitive dye BCECF to evaluate the effect of acute (5-10 min) hypoxia (approximately 3% O(2)) on the regulation of intracellular pH (pH(i)) in astrocyte populations cultured from rat hippocampus. For cells in the nominal absence of CO(2)/HCO(3)(-) at an extracellular pH of 7.40 (37 degrees C), acute hypoxia caused a small (0.05) decrease in steady-state pH(i), but increased the pH(i) recovery rate from an acid load during all but the late phase of the pH(i) recovery. During such pH(i) recoveries, the total acid extrusion rate (phi(E), the product of dpH(i)/dt and proton buffering power) decreased with increasing pH(i). Hypoxia alkali shifted the plot of phi(E) vs. pH(i); over the upper approximately 85% of the phi(E) range, this shift was 0.15-0.30. Hypoxia also stimulated the pH(i) recovery rate from an alkali load. Under normoxic conditions, switching the extracellular buffer to 5% CO(2)/22 mM HCO(-)(3) also alkali shifted the phi(E)-pH(i) plot (upper approximately 85%) by 0.4-0.5. Superimposing hypoxia on CO(2)/HCO(-)(3) further alkali shifted the phi(E)-pH(i) plot (upper approximately 85% of the phi(E) range) by 0.05-0.15. The SITS-insensitive component of phi(E) was alkali shifted by 0.20-0.30, whereas the SITS-sensitive component of phi(E) was depressed in the low pH(i) range. Thus, in the nominal absence of CO(2)/HCO(3)(-), acute hypoxia has little effect on steady-state pH(i) but stimulates acid extrusion and acid loading, whereas in the presence of CO(2)/HCO(-)(3), hypoxia stimulates the SITS-insensitive but inhibits the SITS-sensitive acid extrusion.

Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini.

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na(+)-dependent outward current under voltage-clamp conditions (-60 mV). All three variants had similar external HCO(3)(-) dependencies (apparent K(M) of 4-6 mM) and external Na(+) dependencies (apparent K(M) of 21-36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (-60 mV) expressing NBCe1-A exhibited peak HCO(3)(-)-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current-voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (V(m) = -60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO(3)(-)-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.

Activation of gadolinium-sensitive ion channels in cardiomyocytes in early adaptive stages of volume overload-induced heart failure.

OBJECTIVE: The objective of this study was to investigate whether gadolinium (Gd(3+))-sensitive stretch-activated ion channels (SAC) are basally active in left ventricular (LV) myocytes in early stages of heart failure (HF) induced by volume overload. METHODS: The aortocaval fistula (ACF) model was employed to induce HF due to volume overload in rat. At specific time-points, LV myocytes were acutely isolated using a modified Langendorff apparatus. Whole-cell currents were measured using the patch-clamp technique and intracellular Ca(2+)(Ca(2+)(i)) was examined using fluorescence imaging and the Ca(2+)-sensitive dye Fura-2. RESULTS: Current-voltage data were obtained from sham and ACF myocytes at 5-d and 2-, 6-, 8- and 10-wk post surgery. Compared to data from matching sham rats, a 10 microM Gd(3+)-sensitive current at -100 mV comprised a larger fraction of total current in myocytes from 5-d, 2-wk, and 6-wk ACF rats. In general, the Gd(3+)-sensitive current contributed to inward currents at mV< or =-80 and outward currents at >+20 mV. The enhanced Gd(3+)-sensitive current was absent in myocytes from 8- and 10-wk ACF rats. 10 or 100 microM Gd(3+) had no appreciable effect on resting Ca(2+)(i) of myocytes from 5-d ACF or corresponding sham rats. The Gd(3+)-sensitive current in 5-d ACF myocytes was i) sensitive to the cation-selective SAC inhibitor, GsMTx-4, ii) non-selective for Na(+)/K(+), and iii) impermeable to Ca(2+). CONCLUSION: A basally-active, Gd(3+)- and GsMTx-4-sensitive SAC current that is non-selective for Na(+) and K(+), but impermeable to Ca(2+) under resting conditions is transiently elevated in LV myocytes from rats in early stages of volume overload-induced HF.

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

Regulators of Slc4 bicarbonate transporter activity.

The Slc4 family of transporters is comprised of anion exchangers (AE1-4), Na(+)-coupled bicarbonate transporters (NCBTs) including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2), electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2), and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE), as well as a borate transporter (BTR1). These transporters regulate intracellular pH (pHi) and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO(-) 3 either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO(-) 3 transporter contributes to a cell's ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s) (e.g., Na(+) or Cl(-)). In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both well-known and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family.

Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance.

INTRODUCTION: We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl-) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid. METHODS: Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR(-/-)], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca(2+)]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis. RESULTS: Sinupret-mediated Cl- secretion [ΔISC(µA/cm(2))] was pronounced in WT MNSE (20.7+/-0.9 vs. 5.6+/-0.9(control), p<0.05), CFTR(-/-) MNSE (10.1+/-1.0 vs. 0.9+/-0.3(control), p<0.05) and HSNE (20.7+/-0.3 vs. 6.4+/-0.9(control), p<0.05). The formulation activated Ca(2+) signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl- secretion. Sinupret also enhanced CBF and ASL depth. CONCLUSION: Sinupret stimulates CBF, promotes transepithelial Cl- secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca(2+)-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl- secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.

Inhibition of the Na/bicarbonate cotransporter NBCe1-A by diBAC oxonol dyes relative to niflumic acid and a stilbene.

Na/HCO(3) cotransporters (NBCs) are important regulators of intracellular pH (pH(i) in a variety of organ systems where acid-base status is critical for tissue function. To characterize the pharmacology of NBCs in more detail, we used the two-electrode voltage-clamp technique to examine the effect of previously identified inhibitors of anion exchanger 1 (AE1) on the activity of rat NBCe1-A expressed in Xenopus laevis oocytes. NBC-expressing oocytes voltage-clamped at -60 mV and exposed to a 5% CO(2)/33 mM HCO(3)(-) solution displayed NBC-mediated outward currents that were inhibited by either niflumic acid or one of the two bis-oxonol dyes diBA(3)C4 and diBA(5)C4. NBCe1-A was less sensitive to niflumic acid (apparent K(i) of 100 microM) than 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, apparent K(i) of 36 microM) but more sensitive to the diBAC dyes (apparent K(i) of approximately 10 microM). Based on current-voltage relationships, the diBAC dyes inhibited HCO(3)(-) -induced NBCe1-mediated inward currents more so than outward currents. NBCe1 sensitivity to the dyes was (1) lower in the presence of 40 microM DIDS, (2) unaffected by changes in external HCO(3)(-) concentration and (3) only modestly higher at an external Na(+) concentration of 5, but not 15 or 33, mM. Therefore, the diBAC dyes compete with DIDS but not appreciably with Na(+) or HCO(3)(-) for binding. The mechanism of diBAC inhibition of NBCe1 appears similar to that previously reported for AE1.

Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse.

Tg737(orpk) mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737(orpk) mutants is associated with defects in ion transport, we compared the steady-state pH(i) and Na(+)-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737(orpk) mutant and wild-type mice. The data indicate that Tg737(orpk) mutant choroid plexus epithelium have lower pH(i) and higher Na(+)-dependent HCO(3)(-) transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na(+)-dependent HCO(3)(-) transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737(orpk) mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH(i) and ion transport activity.

A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1.

Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.