A domain mimic increases DeltaF508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (F508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, F508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize F508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant F508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of F508 NBF1. Expression of wild-type NBF1 in the F508 hCFTR cells increased whole cell Cl^– current density to 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a F508/F508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the F508 CFTR protein trafficking defect in cystic fibrosis epithelia. protein processing; mouse; retrovirus; gene therapy     

Innate immune response in CF airway epithelia: hyperinflammatory?

The lack of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the apical membranes of CF airway epithelial cells abolishes cAMP-stimulated anion transport, and bacteria, eventually including Pseudomonas aeruginosa, bind to and accumulate in the mucus. Flagellin released from P. aeruginosa triggers airway epithelial Toll-like receptor 5 and subsequent NF-B signaling and production and release of proinflammatory cytokines that recruit neutrophils to the infected region. This response has been termed hyperinflammatory because so many neutrophils accumulate; a response that damages CF lung tissue. We first review the contradictory data both for and against the idea that epithelial cells exhibit larger-than-normal proinflammatory signaling in CF compared with non-CF cells and then review proposals that might explain how reduced CFTR function could activate such proinflammatory signaling. It is concluded that apparent exaggerated innate immune response of CF airway epithelial cells may have resulted not from direct effects of CFTR on cellular signaling or inflammatory mediator production but from indirect effects resulting from the absence of CFTRs apical membrane channel function. Thus, loss of Cl^–, HCO₃^–, and glutathione secretion may lead to reduced volume and increased acidification and oxidation of the airway surface liquid. These changes concentrate proinflammatory mediators, reduce mucociliary clearance of bacteria and subsequently activate cellular signaling. Loss of apical CFTR will also hyperpolarize basolateral membrane potentials, potentially leading to increases in cytosolic [Ca²⁺], intracellular Ca²⁺, and NF-B signaling. This hyperinflammatory effect of CF on intracellular Ca²⁺ and NF-B signaling would be most prominently expressed during exposure to both P. aeruginosa and also endocrine, paracrine, or nervous agonists that activate Ca²⁺ signaling in the airway epithelia. Pseudomonas aeruginosa; Toll-like receptor; NF-B; oxidative stress; acidic airway surface liquid; calcium     

Unique temperature-activated neurons from pit viper thermosensors

Rattlesnakes, copperheads, and other pit vipers have highly sensitive heat detectors known as pit organs, which are used to sense and strike at prey. However, it is not currently known how temperature change triggers cellular and molecular events that activate neurons supplying the pit organ. We dissociated and cultured neurons from the trigeminal ganglia (TG) innervating the pit organs of the Western Diamondback rattlesnake (Crotalus atrox) and the copperhead (Agkistrodon contortix) to investigate electrophysiological responses to thermal stimuli. Whole cell voltage-clamp recordings indicated that 75% of the TG neurons from C. atrox and 74% of the TG neurons from A. contortix showed a unique temperature-activated inward current (I_T). We also found an I_T-like current in 15% of TG neurons from the common garter snake, a species that does not have a specialized heat-sensing organ. A steep rise in the current-temperature relationship of I_T started just below 18°C, and cooling temperature-responsive TG neurons from 20°C resulted in an outward current, suggesting that I_T is on at relatively low temperatures. Ion substitution and Ca²⁺ imaging experiments indicated that I_T is primarily a monovalent cation current. I_T was not sensitive to capsaicin or amiloride, suggesting that the current did not show similar pharmacology to other mammalian heat-sensitive membrane proteins. Our findings indicate that a novel temperature-sensitive conductance with unique ion permeability and low-temperature threshold is expressed in TG neurons and may be involved in highly sensitive heat detection in snakes. snake; thermosensory; trigeminal; ion conductance     

Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line

The F508 mutationreduces the amount of cystic fibrosis transmembrane conductanceregulator (CFTR) expressed in the plasma membrane of epithelial cells.However, a reduced temperature, butyrate compounds, and "chemicalchaperones" allow F508-CFTR to traffic to the plasma membrane andincrease Cl permeability in heterologous and nonpolarizedcells. Because trafficking is affected by the polarized state ofepithelial cells and is cell-type dependent, our goal was to determinewhether these maneuvers induce F508-CFTR trafficking to the apicalplasma membrane in polarized epithelial cells. To this end, wegenerated and characterized a line of polarized Madin-Darby caninekidney (MDCK) cells stably expressing F508-CFTR tagged with greenfluorescent protein (GFP). A reduced temperature, glycerol, butyrate,or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP(CPT-cAMP)-stimulated transepithelial Cl secretion acrosspolarized monolayers. However, when the basolateral membrane waspermeabilized, butyrate, but not the other experimental maneuvers,increased the CPT-cAMP-stimulated Cl current across theapical plasma membrane. Thus butyrate increased the amount offunctional F508-CFTR in the apical plasma membrane. Butyrate failedto stimulate transepithelial Cl secretion because ofinhibitory effects on Cl uptake across the basolateralmembrane. These observations suggest that studies on heterologous andnonpolarized cells should be interpreted cautiously. The GFP tag onF508-CFTR will allow investigation of F508-CFTR trafficking inliving, polarized MDCK epithelial cells in real time.

     

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte

CLH-3a and CLH-3b are swelling-activated, alternatively spliced Caenorhabditis elegans ClC anion channels that have identical membrane domains but exhibit marked differences in their cytoplasmic NH₂ and COOH termini. The major differences include a 71-amino acid CLH-3a NH₂-terminal extension and a 270-amino acid extension of the CLH-3b COOH terminus. Splice variation gives rise to channels with striking differences in voltage, pH, and Cl^– sensitivity. On the basis of structural and functional insights gained from crystal structures of bacterial ClCs, we suggested previously that these functional differences are due to alternative splicing of the COOH terminus that may change the accessibility and/or function of pore-associated ion-binding sites. We recently identified a mutant worm strain harboring a COOH-terminal deletion mutation in the clh-3 gene. This mutation removes 101 COOH-terminal amino acids unique to CLH-3b and an additional 64 upstream amino acids shared by both channels. CLH-3b is expressed in the worm oocyte, which allowed us to characterize the mutant channel, CLH-3bC, in its native cellular environment. CLH-3bC exhibits altered voltage-dependent gating as well as pH and Cl^– sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn²⁺, prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs. chloride; cell volume; voltage-gated anion channel     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C₂C₁₂ myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24_NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH₂ terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C₂C₁₂ myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C₂C₁₂ myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH₂ terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation. protein-protein interactions; oligomerization; intracellular sorting     

iNOS initiates and sustains metabolic arrest in hypoxic lung adenocarcinoma cells: mechanism of cell survival in solid tumor core

Nitric oxide (NO) modulates cellular metabolism by competitively inhibiting the reduction of O₂ at respiratory complex IV. The aim of this study was to determine whether this effect could enhance cell survival in the hypoxic solid tumor core by inducing a state of metabolic arrest in cancer cells. Mitochondria from human alveolar type II-like adenocarcinoma (A549) cells showed a fourfold increase in NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescence and sixfold increase in Ca²⁺-insensitive NO synthase (NOS) activity during equilibration from PO₂s of 10023 mmHg, which was abolished by N-nitro-L-arginine methyl ester-HCl (L-NAME) and the inducible NOS (iNOS) inhibitor, N⁶-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL). Similarly, cytosolic and compartmented DAF-FM fluorescence increased in intact cells during a transition between ambient PO₂ and 23 mmHg and was abolished by transfection with iNOS antisense oligonucleotides (AS-ODN). In parallel, mitochondrial membrane potential (_m), measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), decreased to a lower steady state in hypoxia without change in glycolytic rate, adenylate energy charge, or cell viability. However, L-NAME or iNOS AS-ODN treatment maintained _m at normoxic levels irrespective of hypoxia and caused a marked activation of glycolysis, destabilization energy charge, and cell death. Comparison with other cancer-derived (H441) or native tissue-derived (human bronchial epithelial; alveolar type II) lung epithelial cells revealed that the hypoxic suppression of _m was common to cells that expressed iNOS. The controlled dissipation of _m, absence of an overt glycolytic activation, and conservation of viability suggest that A549 cells enter a state of metabolic suppression in hypoxia, which inherently depends on the activation of iNOS as PO₂ falls. cancer; oxygen conformation; mitochondrial nitric oxide synthase; mitochondrial metabolism     

Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca²⁺-sensitive K⁺ (mtK_Ca) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK_Ca channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K⁺ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (_m). To test this hypothesis, we measured effects of NS-1619, a putative mtK_Ca channel opener, and valinomycin, a K⁺ ionophore, on mitochondrial respiration, _m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O₂·min^–1·mg protein^–1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O₂·min^–1·mg protein^–1, respectively, with the FADH₂-linked substrate succinate (+ rotenone); these effects were abolished by the mtK_Ca channel blocker paxilline. _m was not decreased by 10–30 µM NS-1619 with either substrate, but H₂O₂ release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H₂O₂ release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained _m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K⁺ influx during states 2 and 4 increases mitochondrial respiration while maintaining _m; this allows singlet electron uptake by O₂ and ROS generation. mitochondrial bioenergetics; heart mitochondria     

Human cytomegalovirus-induced host cell enlargement is iron dependent

A hallmark of human cytomegalovirus (HCMV) infection is the characteristic enlargement of the host cells (i.e., cytomegaly). Because iron (Fe) is required for cell growth and Fe chelators inhibit viral replication, we investigated the effects of HCMV infection on Fe homeostasis in MRC-5 fibroblasts. Using the metallosensitive fluorophore calcein and the Fe chelator salicylaldehyde isonicotinoyl hydrazone (SIH), the labile iron pool (LIP) in mock-infected cells was determined to be 1.04 ± 0.05 µM. Twenty-four hours postinfection (hpi), the size of the LIP had nearly doubled. Because cytomegaly occurs between 24 and 96 hpi, access to this larger LIP could be expected to facilitate enlargement to 375% of the initial cell size. The ability of Fe chelation by 100 µM SIH to limit enlargement to 180% confirms that the LIP plays a major role in cytomegaly. The effect of SIH chelation on the mitochondrial membrane potential (_M) and morphology was studied using the mitochondrial voltage-sensitive dye JC-1. The mitochondria in mock-infected cells were heterogeneous with a broad distribution of _M and were threadlike. In contrast, the mitochondria of HCMV-infected cells had a more depolarized _M distributed over a narrow range and were grainlike in appearance. However, the HCMV-induced alteration in _M was not affected by SIH chelation. We conclude that the development of cytomegaly is inhibited by Fe chelation and may be facilitated by an HCMV-induced increase in the LIP. cell size; mitochondria     

Adaptation of the Euryhaline Charophyte Lamprothamnium papulosum to Brackish and Freshwater: Turgor Pressure and Vacuolar Solute Concentrations During Steady-State Culture and After Hypo-osmotic Treatment

The euryhaline charophyte Lamprothamnium papulosum (Wallr.)J. Gr. was adapted to media with decreasing salinities rangingfrom 550 to 0 mosmol kg^–1. Vegetative plants grown inmedia with osmotic pressures (⁰) in the range of 550 to 130mosmol kg^–1 maintained a constant turgor pressure () at309 + 7 mosmol kg^–1. The ions K+, Na+ and Cl–, werethe predominant solutes in the vacuole. Changes in their concentrationsaccount for the variation in internal osmotic pressure (¹) with,⁰. The divalent ions Mg²⁺, Ca²⁺ and were also present in significant amounts, but their concentrationsdid not alter with changes in, ⁰. In cells subjected to hypo-osmotic shock the regulation of was incomplete. The turgor pressure increased from 302 to 383mosmol kg^–1. The first rapid response to the sudden decreasein ⁰ was a loss of K⁺ and Cl^–. In contrast to the decreasein ionic concentrations an accumulation of sucrose occurredwhich could account for the increase of . The increase in sucroseconcentration started 24 to 48 h after the downshock and reachedits highest value after 3 to 4 weeks. The sucrose concentrationin the vacuole was up to 320 mol m^–3. During this timethe ionic content continued to decrease but did not counterbalancethe sucrose concentration sufficiently to regain the original. High sucrose levels accompanied by an enhanced were also observedduring the period of fructification (sexual reproduction: formationof antheridia and oogonia) in Lamprothamnium kept under conditionsof constant salinity. It is concluded that high sucrose content and elevated arecharacteristic of sexual reproduction in this charophyte. Lamprothamniumis able to tolerate different during various developmentalstages (e.g. vegetative and reproductive phases). Key words: Lamprothamnium papulosum, sucrose, turgor pressure     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Protein kinase C mediates the inhibitory effect of substance P on HCO3- secretion from guinea pig pancreatic ducts

The inhibitory control of pancreatic ductal HCO₃^– secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO₃^– secretion by modulating a Cl^–-dependent HCO₃^– efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268–C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO₃^– secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO₃^– secretion by 40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 µM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO₃^– secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the -, _I-, -, -, -, -, -, and µ-isoforms of PKC. In microperfused ducts, luminal H₂DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin-stimulated HCO₃^– secretion. SP did not inhibit secretion further when H₂DIDS was present in the lumen, suggesting that SP and H₂DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell. pancreas; Cl^–/HCO₃^– exchanger; inhibition; epithelium     

Spontaneous mitochondrial depolarizations are independent of SR Ca2+ release

The mitochondrial membrane potential (_m) underlies many mitochondrial functions, including Ca²⁺ influx into the mitochondria, which allows them to serve as buffers of intracellular Ca²⁺. Spontaneous depolarizations of _m, flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca²⁺ sparks. We have shown previously that an increase in global Ca²⁺ in smooth muscle cells causes an increase in mitochondrial Ca²⁺ and depolarization of _m. Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca²⁺ released focally in Ca²⁺ sparks. High-speed three-dimensional imaging was used to monitor _m in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca²⁺ not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca²⁺ release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca²⁺ release from the SR does not cause flickers in the cells employed here. mitochondria; mitochondrial membrane potential; intracellular calcium; permeability transition pore; sarcoplasmic reticulum     

Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl(-) secretion by T84 cells

Wortmannin is a potent inhibitor ofphosphatidylinositol 3-kinase (PI3K) and membrane trafficking in manycells. To test the hypothesis that cystic fibrosis transmembraneconductance regulator (CFTR) traffics into and out of the plasmamembrane during cAMP-stimulated epithelial Clsecretion, we have studied the effects of wortmannin onforskolin-stimulated Cl secretion by the humancolonic cell line T84. At the PI3K inhibitory concentration of 100 nM,wortmannin did not affect significantly forskolin-stimulatedCl secretion measured as short-circuit current(I_SC). However, 500 nM wortmannin significantlyinhibited forskolin-stimulated I_SC. cAMP activationof apical membrane CFTR Cl channels in-toxin-permeabilized monolayers was not reduced by 500 nMwortmannin, suggesting that inhibition of other transporters accountsfor the observed reduction in T84 Cl secretion.Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP),but wortmannin did not alter forskolin inhibition of apical HRPendocytosis. In the absence of forskolin, wortmannin stimulated HRPendocytosis significantly. We conclude that, in T84 cells, apical fluidphase endocytosis is not dependent on PI3K activity and that CFTR doesnot recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.

     

Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production

Recently, we demonstrated that the peroxisome proliferator-activated receptor- (PPAR-) ligands, either 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) or ciglitazone, increased endothelial nitric oxide (·NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR--stimulated endothelial·NO release remain to be defined. Superoxide anion radical (O₂^–·) combines with ·NO to decrease·NO bioavailability. NADPH oxidase, which produces O₂^–·, and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O₂^–·, thereby contribute to regulation of endothelial cell·NO metabolism. Therefore, we examined the ability of PPAR- ligands to modulate endothelial O₂^–· metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 µM 15d-PGJ₂ or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O₂^–· production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ₂ or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91^phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ₂ or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR- ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial·NO production, PPAR- ligands enhance endothelial·NO bioavailability, in part by altering endothelial O₂^–· metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR- ligands directly alter vascular endothelial function. reduced nicotinamide adenine dinucleotide phosphate oxidase; copper/zinc superoxide dismutase; nitric oxide; endothelial cells     

The prolyl hydroxylase oxygen-sensing pathway is cytoprotective and allows maintenance of mitochondrial membrane potential during metabolic inhibition

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (_mito) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, _mito is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse _mito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of _mito in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F₁F₀-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult. fumarate; hibernation; cardioprotection; anaplerotic     

Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak

_v5-Integrin is the sole integrin receptor at the retinal pigment epithelium (RPE)-photoreceptor interface and promotes RPE phagocytic signaling to the tyrosine kinase Mer tyrosine kinase (MerTK) once a day in response to circadian photoreceptor shedding. Herein we identify a novel role for _v5-integrin in permanent RPE-photoreceptor adhesion that is independent of _v5's function in retinal phagocytosis. To compare retinal adhesion of wild-type and ₅-integrin^–/– mice, we mechanically separated RPE and neural retina and quantified RPE protein and pigment retention with the neural retina. Lack of _v5-integrin with normal expression of other RPE integrins greatly weakened retinal adhesion in young mice and accelerated its age-dependent decline. Unexpectedly, the strength of wild-type retinal adhesion varied with a diurnal rhythm that peaked 3.5 h after light onset, after the completion of phagocytosis, when integrin signaling to MerTK is minimal. Permanent _v5 receptor deficiency attenuated the diurnal peak of retinal adhesion in ₅-integrin^–/– mice. These results identify _v5-integrin as the first RPE receptor that contributes to retinal adhesion, a vital mechanism for long-term photoreceptor function and viability. Furthermore, they indicate that _v5 receptors at the same apical plasma membrane domain of RPE cells fulfill two separate functions that are synchronized by different diurnal rhythms. circadian rhythm; knockout; photoreceptors; retinal pigment epithelium     

A comparison of the impacts of various nitrogen sources on acid-base balance in C3 Triticum aestivum L. and C4 Zea mays L. plants

This work aimed to study the impacts of acquisition and assimilationof various nitrogen sources, i.e. NO₃^–, NH₄⁺ or NH₄NO₃,in combination with gaseous NH₃ on plant growth and acid-basebalance in higher plants. Plants of C₃ Triticum aestivum L.and C₄ Zea mays L. grown with shoots in ambient air in hydroponicculture solutions with 2 mol m^–3 of nitrogen source asNO₃^–, NH₄⁺ or NH₄NO₃ for 21 d and 18 d, respectively,had their shoots exposed either to 320 µg m^–3 NH₃or to ambient air for 7 d. Variations in plant growth (leaves,stubble and roots), and OH^– and H⁺ extrusions as wellas the relative increases in nitrogen, carbon and carboxylatewere determined. These data were computed as H⁺/N, H⁺/C, (C-A)/N,and (C-A)/C to analyse influences of different nitrogen sourceson acid-base balance in C₃ Triticum aestivum and C₄ Zea maysplants. Root growth in dry weight gain was significantly reduced bytreatment with 320 µg m^–3 NH₃ in Triticum aestivumand Zea mays growing with different N-forms, whereas leaf growthwas not significantly affected by NH₃. In comparison with C₃Triticum aestivum, non-fumigated C₄ Zea mays had low ratiosof OH^–/N in NO₃^–3-grown plants and of H⁺/N in NH₄⁺- and NH ₄NO₃-grown plants. Utilization of NH₃ from the atmospherereduced both the OH^–N ratios in NO₃^– -grown plantsand the H⁺/N ratio in NH₄⁺ - and NH₄NO₃ -grown plants of bothspecies. Furthermore, Zea mays had higher ratios of (C-A)/Nin NH₄⁺ - and NH₄NO₃-grown plants than Triticum aestivum. Thismeans that C₄ Zea mays had synthesized more organic anion perunit increase in organic N than C₃ Triticum aestivum plants.Within both species, different nitrogen sources altered theratios of (C-A)/N in the order: NH₄NO₃>NH₄⁺>NO₃^–.Fumigation with NH₃ increased organic acid synthesis in NO₃^–- and NH₄⁺ - grown plants of Triticum aestivum, whereas it decreasedorganic acid synthesis in Zea mays plants under the same conditions.Furthermore, these differences in acid-base regulation betweenC₃ Triticum aestivum and C₄ Zea mays plants growing with differentnitrogen sources are discussed. Key words: Acid-base balance, ammonia, ammonium, nitrate, ammonium nitrate, C₃ Triticum aestivum L., C₄ Zea mays L.