Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), F508, leads to the absence of CFTR Cl^– channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of F508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl^– channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of F508-CFTR. cystic fibrosis     

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     

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     

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     

Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons

When neurons in culture are transiently stressed by inhibition of ATP synthesis, they rapidly form within their neurites rodlike actin inclusions that disappear when the insult is removed. Oxidative stress, excitotoxic insults, and amyloid -peptide oligomers also induce rods. Immunostaining of neurites indicates that these rods also contain the majority of the actin filament dynamizing proteins, actin-depolymerizing factor (ADF) and cofilin (AC). If the rods reappear within 24 h after the stress is removed, the neurite degenerates distal to the rod but with no increase in neuronal death. Here, rods were generated in cultured rat E18 hippocampal cells by overexpression of a green fluorescent protein chimera of AC. Surprisingly, we have found that, for a short period (60 min) immediately after initial rod formation, the loss of mitochondrial membrane potential (_m) and ATP in neurites with rods is slower than in neurites without them. The _m was monitored with the fluorescent dye tetramethylrhodamine methyl ester, and ATP was monitored with the fluorescent ion indicator mag-fura 2. Actin in rods is less dynamic than is filamentous actin in other cytoskeletal structures. Because _m depends on cellular ATP and because ATP hydrolysis associated with actin filament turnover is responsible for a large fraction of neuronal energy consumption (50%), the formation of rods transiently protects neurites by slowing filament turnover and its associated ATP hydrolysis. actin dynamics; neurodegeneration; actin inclusions; neuroprotection; ischemia     

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     

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     

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta

Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell     

PKC zeta participates in activation of inflammatory response induced by enteropathogenic E. coli

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-B activation but that other signaling molecules such as PKC may be involved. The aim of this study was to determine whether PKC is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC pseudosubstrate (MYR-PKC-PS), or transient expression of a nonfunctional PKC significantly suppressed EPEC-induced IB phosphorylation. Although PKC can activate ERK, MYR-PKC-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC can regulate NF-B activation by interacting with and activating IB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC-IKK complexes from infected intestinal epithelial cells and recombinant IB as a substrate showed a 2.5-fold increase in IB phosphorylation. PKC can also regulate NF-B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC-PS, suggesting that other signaling events may be involved in this particular arm of NF-B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC and ERK that lead to NF-B activation, thus ensuring the proinflammatory response. inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C; IB kinase; extracellular signal-regulated kinase     

Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (, I, II, and ), novel [, , , and µ (also known as PKD),], and atypical ( and /), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms , I, and and complete translocation of PKC and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKC and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms , I, and were dispensable with regard to these same phenomena. signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium     

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     

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     

Direct association of RhoA with specific domains of PKC-alpha

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)- and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC- involved in direct interaction with RhoA, His-tagged PKC- proteins of individual domains and different combinations of PKC- domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC- in this study. The data indicate that RhoA directly bound to full-length PKC-, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC- [PKC- (C1)] (70.48 ± 20.78% above control), PKC- (C2) (72.26 ± 29.96% above control), and PKC- (C4) (90.58 ± 26.79% above control), but not to PKC- (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC- (C1, C2, and C3) or to PKC- (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC- (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC- (C2, C3, and C4) and PKC- (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-. The studies using cells transfected with truncated forms of PKC- indicate that PKC- (C1 and C2), PKC- (C1, C2, and C3), and PKC- (C2 and C3) did not associate with RhoA. Only full-length PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC- with RhoA may require the C4 domain. domains; histidine; fusion proteins     

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     

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     

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     

Salutary effects of 17beta-estradiol on T-cell signaling and cytokine production after trauma-hemorrhage are mediated primarily via estrogen receptor-alpha

Although 17-estradiol (E2) administration following trauma-hemorrhage prevents the suppression in splenocyte cytokine production, it remains unknown whether the salutary effects of 17-estradiol are mediated via estrogen receptor (ER)- or ER-. Moreover, it is unknown which signaling pathways are involved in 17-estradiol's salutary effects. Utilizing an ER-- or ER--specific agonist, we examined the role of ER- and ER- in E2-mediated restoration of T-cell cytokine production following trauma-hemorrhage. Moreover, since MAPK, NF-B, and activator protein (AP)-1 are known to regulate T-cell cytokine production, we also examined the activation of MAPK, NF-B, and AP-1. Male rats underwent trauma-hemorrhage (mean arterial pressure 40 mmHg for 90 min) and fluid resuscitation. ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic T cells were isolated, and their IL-2 and IFN- production and MAPK, NF-B, and AP-1 activation were measured. T-cell IL-2 and IFN- production was decreased following trauma-hemorrhage, and this was accompanied with a decrease in T-cell MAPK, NF-B, and AP-1 activation. PPT or 17-estradiol administration following trauma-hemorrhage normalized those parameters, while DPN administration had no effect. Since PPT, but not DPN, administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the T-cell suppression, it appears that ER- plays a predominant role in mediating the salutary effects of 17-estradiol on T cells following trauma-hemorrhage, and that such effects are likely mediated via normalization of MAPK, NF-B, and AP-1 signaling pathways. shock; MAPK; NF-B; activator protein-1; propyl pyrazole triol; diarylpropionitrile     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

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     

Stretch increases alveolar epithelial permeability to uncharged micromolecules

We measured stretch-induced changes in transepithelial permeability in vitro to uncharged tracers 1.5–5.5 Å in radius to identify a critical stretch threshold associated with failure of the alveolar epithelial transport barrier. Cultured alveolar epithelial cells were subjected to a uniform cyclic (0.25 Hz) biaxial 12, 25, or 37% change in surface area (SA) for 1 h. Additional cells served as unstretched controls. Only 37% SA (100% total lung capacity) produced a significant increase in transepithelial tracer permeability, with the largest increases for bigger tracers. Using the permeability data, we modeled the epithelial permeability in each group as a population of small pores punctuated by occasional large pores. After 37% SA, increases in paracellular transport were correlated with increases in the radii of both pore populations. Inhibition of protein kinase C and tyrosine kinase activity during stretch did not affect the permeability of stretched cells. In contrast, chelating intracellular calcium and/or stabilizing F-actin during 37% SA stretch reduced but did not eliminate the stretch-induced increase in paracellular permeability. These results provide the first in vitro evidence that large magnitudes of stretch increase paracellular transport of micromolecules across the alveolar epithelium, partially mediated by intracellular signaling pathways. Our monolayer data are supported by whole lung permeability results, which also show an increase in alveolar permeability at high inflation volumes (20 ml/kg) at the same rate for both healthy and septic lungs. ventilator-induced lung injury; acute lung injury; barrier properties