Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming _1A-subunits of human P/Q-type Ca²⁺ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human _1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of _1A-WT with each _1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba²⁺ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of _1A-mutants increased, suggesting the presence of an _1A-mutant-specific suppression effect. When we coexpressed _1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of _1A-WT and mutant using a splice variant of _1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-_1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects. channelopathy; voltage clamp; Xenopus oocytes; cerebellum; splice variants     

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     

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     

Protein kinase Cdelta-dependent and -independent signaling in genotoxic response to treatment of desferroxamine, a hypoxia-mimetic agent

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKC is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKC by RNAi (sh-PKC) or expression of the kinase-dead (Lys376Arg) mutant of PKC (PKCKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (-H2AX), a hallmark for DNA damage, was altered by sh-PKC, and sh-PKC completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKC-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKC and caspase-3, suggesting that PKCKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKC-dependent and -independent manners. In summary, these findings suggest that the activation of a PKC-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment. DNA damage; caspase-3; Akt     

Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling

Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) _1S-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of _1S (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (_1S null) myotubes. Compared with wild-type _1S, caffeine-activated Ca²⁺ release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. Although maximal voltage-gated Ca²⁺ release was similar in _1S- and R1086H-expressing myotubes, the voltage dependence of Ca²⁺ release was shifted 5 mV to more negative potentials in R1086H-expressing myotubes. Our results demonstrate that _1S functions as a negative allosteric modulator of release channel activation by caffeine/voltage and that the R1086H MH mutation in the intracellular III-IV linker disrupts this negative regulatory influence. Moreover, a low caffeine concentration (2 mM) caused a similar shift in voltage dependence of Ca²⁺ release in _1S- and R1086H-expressing myotubes. Compared with _1S-expressing myotubes, maximal L channel conductance (G_max) was reduced in R1086H-expressing myotubes (_1S 130 ± 10.2, R1086H 88 ± 6.8 nS/nF; P < 0.05). The decrease in G_max did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (G_max/Q_max) was similar in _1S- and R1086H-expressing myotubes and a similar decrease in G_max was observed for an analogous mutation engineered into the cardiac L channel (R1217H). In addition, both R1086H and R1217H DHPRs targeted normally and colocalized with RyR1 in sarcoplasmic reticulum (SR)-sarcolemmal junctions. These results indicate that the R1086H MH mutation in _1S enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators. excitation-contraction coupling; calcium channel; muscle disease     

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF- plus cycloheximide (TNF-/CHX). Specific inhibition of NF-B by the recombinant adenoviral vector containing the IB superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-B activity and increased the sensitivity to TNF-/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-B activity and resulted in increased resistance to TNF-/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-/CHX-induced apoptosis, at least partially, through the activation of NF-B signaling in IECs. polyamines; -difluoromethylornithine; X-linked inhibitor of apoptosis protein; IB     

Effects of p38MAPK isoforms on renal mesangial cell inducible nitric oxide synthase expression

Several related isoforms of p38^MAPK have been identified and cloned in many species. Although they all contain the dual phosphorylation motif TGY, the expression of these isoforms is not ubiquitous. p38 and -2 are ubiquitously expressed, whereas p38 and - appear to have more restricted expression. Because there is evidence for selective activation by upstream kinases and selective preference for downstream substrates, the functions of these conserved proteins is still incompletely understood. We have demonstrated that the renal mesangial cell expresses the mRNA for all the isoforms of p38^MAPK, with p38 mRNA expressed at the highest level, followed by p38 and the lowest levels of expression by p382 and -. To determine the functional effects of these proteins on interleukin (IL)-1-induced inducible nitric oxide synthase (iNOS) expression, we transduced TAT-p38 chimeric proteins into renal mesangial cells and assessed the effects of wild-type and mutant p38 isoforms on ligand induced iNOS expression. We show that whereas p38 and - had minimal effects on iNOS expression, p38 and -2 significantly altered its expression. p38 mutant and p382 wild-type dose dependently inhibited IL-1-induced iNOS expression. These data suggest that p38 and 2 have reciprocal effects on iNOS expression in the mesangial cell, and these observations may have important consequences for the development of selective inhibitors targeting the p38^MAPK family of proteins. TAT proteins; p38 MAPK; inducible nitric oxide synthase; mesangial cell; interleukin-1     

Activation of PLC-delta1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)- activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca²⁺ stimulated an eightfold increase in PLC-1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G_i/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen²,D-Pen⁵]enkephalin, and A₁ agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca²⁺ influx and was not observed in the absence of extracellular Ca²⁺, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca²⁺ channels. Treatment of the cells with a G_q/13-coupled receptor agonist, CCK-8, caused only transient, PLC-1-mediated PI hydrolysis. Unlike G_i/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G₁₃ minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca²⁺ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (25%) and strongly inhibited by SKF-96365 (75%) and in cells expressing PLC-1(E341R/D343R). Agonist-independent Ca²⁺ release or Ca²⁺ influx via voltage-gated Ca²⁺ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-1 antibody or nifedipine. We conclude that PLC-1 is activated by G_i/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca²⁺ influx via store-operated Ca²⁺ channels. phospholipase C; G protein     

Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants

Fabry disease is a lysosomal storage disorder caused by deficiency of -galactosidase A (-Gal A) resulting in lysosomal accumulation of glycosphingolipid globotriosylceramide Gb3. Misfolded -Gal A variants can have residual enzyme activity but are unstable. Their lysosomal trafficking is impaired because they are retained in the endoplasmic reticulum (ER) by quality control. Subinhibitory doses of the competitive inhibitor of -Gal A, 1-deoxygalactonojirimycin (DGJ), stabilize mutant -Gal A in vitro and correct the trafficking defect. We showed by immunolabeling that the chaperone-like action of DGJ significantly reduces the lysosomal Gb3 storage in human Fabry fibroblasts harboring the novel mutations T194I and V390fsX8. The specificity of the DGJ effect was proven by RNA interference. Electron microscopic morphometry demonstrated a reduction of large-size, disease-associated lysosomes and loss of characteristic multilamellar lysosomal inclusions on DGJ treatment. In addition, the pre-Golgi intermediates were decreased. However, the rough ER was not different between DGJ-treated and untreated cells. Pulse-chase experiments revealed that DGJ treatment resulted in maturation and stabilization of mutant -Gal A. Genes involved in cell stress signaling, heat shock response, unfolded protein response, and ER-associated degradation show no apparent difference in expression between untreated and DGJ-treated fibroblasts. The DGJ treatment has no apparent cytotoxic effects. Thus our data show the usefulness of a pharmacological chaperone for correction of the lysosomal storage in Fabry fibroblasts harboring different mutations with residual enzyme activity. Pharmacological chaperones acting on misfolded, unstable mutant proteins that exhibit residual biological activity offer a convenient and cost-efficient therapeutic strategy. protein trafficking; Gb3 storage; lysosomes     

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     

Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3

Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C.     

The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with beta-arrestins

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with -arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of -arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only -arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with -arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca²⁺ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with -arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with -arrestin 1 and determine the rate of resensitization. desensitization; endocytosis; tachykinins     

Dominant-negative PKC-epsilon impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini

We investigated the involvement of PKC- in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC- cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 µM, 2–15 min) significantly (P 0.05) increased PKC- recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC- association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC- in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and -hexosaminidase. The chemical inhibitor GF-109203X (10 µM, 3 h), which inhibits PKC-, -, -, and -, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 µM, 3 h), which inhibits only PKC- and -. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC- significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC- transduction suppressed its carbachol-stimulated release. We propose that DN-PKC- alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis. lacrimal gland; acinar epithelial cell; exocytosis; polymeric immunoglobulin A receptor     

A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse

The actin-binding protein -actinin-3 is one of the two isoforms of -actinin that are found in the Z-discs of skeletal muscle. -Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of -actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of -actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, -actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of -actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed -actinin-3 knockout (KO) mouse. -Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of -actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. -Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with -actinin-3-positive muscles, -actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that -actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from -actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance. extensor digitorum longus     

Innate immune responses of human tracheal epithelium to Pseudomonas aeruginosa flagellin, TNF-alpha, and IL-1beta

We measured innate immune responses by primary human tracheal epithelial (HTE) cells grown as confluent, pseudostratified layers during exposure to inflammatory activators on apical vs. basolateral surfaces. Apical Pseudomonas aeruginosa strain PAK (but not flagellin mutant PAK·fliC), flagellin, and flagellin + PAK·fliC activated NF-B and IL-8 expression and secretion. In contrast, HTE cells were insensitive to LPS compared to flagellin. Flagellin activated NF-B in columnar but not basal cells. IL-1 + TNF- elicited responses similar to those of flagellin. Basolateral flagellin or IL-1 + TNF- caused 1.5- to 4-fold larger responses, consistent with the fact that NF-B activation occurred in both columnar and basal cells. MyD88 (toll receptor-associated adapter), IL-1 receptor (IL1R)1, and TNF- receptor (TNFR)1 were expressed in columnar and basal cells. ZO-1 was localized to tight junctions of columnar cells but not to basal cells. We infer the following. 1) Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and MyD88, often associated with flagellin-activated cell signaling. 2) IL-1 + TNF- in the ASL also activate columnar cells, and these cells also express IL1R1 and TNFR1. 3) Apical flagellin, IL-1, and TNF- do not activate basal cells because tight junctions between columnar cells prevent access from the apical surface to the basal cells. 4) Exposure of basolateral surfaces to inflammatory activators elicits larger responses because both columnar and basal cells are activated, likely because both cell types express receptors for flagellin, IL-1, and TNF-. toll-like receptor; nuclear factor-B; interleukin-8; tumor necrosis factor; interleukin-1     

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     

The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase and activate ERK, G_q to stimulate phospholipase C and phospholipase A₂, and G to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G_12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G_i and G_q, and a p115RhoGEF construct containing the RGS domain for G_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT inhibited extracellular Ca²⁺-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G_i and G_q. This suggests that the CaR may regulate cytoskeleton via G_12/13, Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins     

Modulation of vascular smooth muscle cell migration by calcium/ calmodulin-dependent protein kinase II-delta 2

Previous studies demonstrated a requirement for multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (₂ or _C) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII₂ were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr¹⁷, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKII₂ inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKII₂ autophosphorylation on Thr²⁸⁷ after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII₂ phosphorylated substrate in vitro without added Ca²⁺/calmodulin and in the intact cell without added Ca²⁺-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKII₂ on Thr²⁸⁷. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII₂, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII₂ mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII₂ isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII₂ autophosphorylation may play an important role in PDGF-stimulated VSM cell migration. calcium/calmodulin-dependent protein kinase II; cell migration; adenovirus; autophosphorylation; chemotaxis; platelet-derived growth factor     

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     

Neuropeptide substance P upregulates chemokine and chemokine receptor expression in primary mouse neutrophils

Neuropeptides play an important role in the active communication between the nervous and immune systems. Substance P (SP) is a prominent neuropeptide involved in neurogenic inflammation and has been reported to exert various proinflammatory actions on inflammatory leukocytes including neutrophils. The present study further investigated the modulatory effect of SP (1 µM) on chemokine production and chemokine receptor expression in primary mouse neutrophils. Our results showed that SP primed neutrophils for chemotactic responses not only to the CXC chemokine macrophage inflammatory protein (MIP)-2/CXCL2 but also to the CC chemokine MIP-1/CCL3. The activating effect of SP on neutrophils was further evidenced by upregulation of the CD11b integrin, the activation marker of neutrophils. SP induced both the mRNA and protein expression of the chemokines MIP-1/CCL3 and MIP-2/CXCL2 in neutrophils and upregulated the chemokine receptors CC chemokine receptor (CCR)-1 and CXC chemokine receptor (CXCR)-2. This stimulatory effect on chemokine and chemokine receptor expression in neutrophils was further found to be neurokinin-1 receptor (NK-1R) specific. Pretreatment with selective NK-1R antagonists inhibited SP-triggered activation of neutrophils and chemokine and chemokine receptor upregulation. Moreover, SP-induced chemokine upregulation was NF-B dependent. SP time dependently induced NF-B p65 binding activity, IB degradation, and NF-B p65 nuclear translocation in neutrophils. Inhibition of NF-B activation with its inhibitor Bay11-7082 (10 µM) abolished SP-induced NF-B binding activity and upregulation of MIP-1/CCL3 and MIP-2/CXCL2 in neutrophils. Together, these results suggest that SP exerts a direct stimulatory effect on the expression of chemokines and chemokine receptors in mouse neutrophils. The effect is NK-1R mediated, involving NF-B activation. chemokines and receptors; neuro-immune interaction; neurokinin-1 receptor; primary leukocytes; NF-B activation