bluuebunny/crossref_metadata_2025 huggingface dataset:
Skeletal muscle sarcoplasmic reticulum glycogen status influences Ca2+ uptake supported by endogenously synthesized ATP
Authors: Lees Simon J. , Williams Jay H.
Published: January 2004

The purpose of this investigation was to determine whether there is a link between sarcoplasmic reticulum (SR) glycogen status and SR Ca2+ handling. In this investigation, skeletal muscle SR was purified from female Sprague-Dawley rats (200–250 g). Glycogen was extracted from the SR purified from one hindlimb, whereas the SR purified from the contralateral limb served as control. Before removal of the tissue, the animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Both α-amylase treatment (AM) and removal of EDTA from the homogenization and storage buffers reduced the amount of glycogen associated with the SR ( P < 0.05). AM treatment reduced the glycogen phosphorylase content of SR ( P < 0.05). In contrast, creatine kinase (CK) and pyruvate kinase (PK) contents were increased after both glycogen extraction protocols ( P < 0.05). Under exogenous ATP conditions, both AM and EDTA-free (EF) treatments resulted in an increase in Ca2+-stimulated ATPase activity when normalized to sarco(endo)plasmic reticulum calcium-ATPase (SERCA) content ( P < 0.05). CK and PK-supported SR Ca2+ uptake was decreased ( P < 0.05) in the AM group when normalized to SERCA and CK or SERCA and PK content, respectively. AM was more effective than the EF for extracting glycogen associated with purified SR. Glycogen extraction alters the yield of purified SR proteins and must be taken into account when investigating SR calcium handling. Removal of glycogen from purified SR causes a change in Ca2+-handling properties as measured by ATPase and uptake activities.
Ca transients from Ca channel activity in rat cardiac myocytes reveal dynamics of dyad cleft and troponin C Ca binding
Authors: Vadakkadath Meethal Sivan , Potter Katherine T. , Redon David , Heisey Dennis M. , Haworth Robert A.
Published: January 2004

The properties of the dyad cleft can in principle significantly impact excitation-contraction coupling, but these properties are not easily amenable to experimental investigation. We simultaneously measured the time course of the rise in integrated Ca current ( ICa) and the rise in concentration of fura 2 with Ca bound ([Ca-fura 2]) with high time resolution in rat myocytes for conditions under which Ca entry is only via L-type Ca channels and sarcoplasmic reticulum (SR) Ca release is blocked, and compared these measurements with predictions from a finite-element model of cellular Ca diffusion. We found that 1) the time course of the rise of [Ca-fura 2] follows the time course of integrated ICa plus a brief delay (1.36 ± 0.43 ms, n = 6 cells); 2) from the model, high-affinity Ca binding sites in the dyad cleft at the level previously envisioned would result in a much greater delay (≥3 ms) and are therefore unlikely to be present at that level; 3) including ATP in the model promoted Ca efflux from the dyad cleft by a factor of 1.57 when low-affinity cleft Ca binding sites were present; 4) the data could only be fit to the model if myofibrillar troponin C (TnC) Ca binding were low affinity (4.56 μM), like that of soluble troponin C, instead of the high-affinity value usually used (0.38 μM). In a “good model,” the rate constants for Ca binding and dissociation were 0.375 times the values for soluble TnC; and 5) consequently, intracellular Ca buffering at the rise of the Ca transient is inferred to be low.
Ca2+ influx through α1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle
Authors: Shtifman Alexander , Paolini Cecilia , López José R. , Allen Paul D. , Protasi Feliciano
Published: January 2004

Differentiated primary myotubes isolated from wild-type mice exhibit ryanodine-sensitive, spontaneous global Ca2+ oscillations as well as spontaneous depolarizations in the plasma membrane. Immunolabeling of these myotubes showed expression of both α1S dihydropyridine receptors (DHPRs) and ryanodine-sensitive Ca2+-release channel 1 (RyR1), the two key proteins in skeletal excitation-contraction (E-C) coupling. Spontaneous global Ca2+ oscillations could be inhibited by addition of 0.1 mM CdCl2/0.5 mM LaCl3 or 5 μM nifedipine to the extracellular bathing solution. After either treatment, Ca2+ oscillations could be restored upon extensive washing. Although exposure to DHPR antagonists completely blocked Ca2+ oscillations, normal orthograde signaling between DHPRs and RyRs, such as that elicited by 80 mM KCl depolarization, was still observed. In addition, we showed that spontaneous Ca2+ oscillations were never present in cultured mdg myotubes, which lack the expression of α1SDHPRs. These results suggest that under physiological conditions in conjunction with the mechanical coupling between the α1SDHPRs and RyR1, the initiation of Ca2+ oscillations in myotubes may be facilitated, in part, by the Ca2+ influx through the α1s-subunit of the DHPR.
IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells
Authors: Oka Tatsuya , Hori Masatoshi , Tanaka Akane , Matsuda Hiroshi , Karaki Hideaki , Ozaki Hiroshi
Published: January 2004

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcϵRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca2+ level ([Ca2+]i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca2+]i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca2+]i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca2+]i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca2+]i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca2+ signaling and degranulation.
Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury
Authors: Lovering Richard M. , De Deyne Patrick G.
Published: January 2004

The purpose of this study was to evaluate the integrity of the muscle membrane and its associated cytoskeleton after a contraction-induced injury. A single eccentric contraction was performed in vivo on the tibialis anterior (TA) of male Sprague-Dawley rats at 900°/s throughout a 90°-arc of motion. Maximal tetanic tension (Po) of the TAs was assessed immediately and at 3, 7, and 21 days after the injury. To evaluate sarcolemmal integrity, we used an Evans blue dye (EBD) assay, and to assess structural changes, we used immunofluorescent labeling with antibodies against contractile (myosin, actin), cytoskeletal (α-actinin, desmin, dystrophin, β-spectrin), integral membrane (α- and β-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P0 was significantly reduced to 4.23 ± 0.22 N, compared with 8.24 ± 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 ± 22% of fibers from the injured TA, compared with 0% in noninjured controls. We found a significant association between EBD-positive fibers and the loss of complete dystrophin labeling. The loss of dystrophin was notable because organization of other components of the subsarcolemmal cytoskeleton was affected minimally (β-spectrin) or not at all (α- and β-dystroglycan). Labeling with specific antibodies indicated that dystrophin's COOH terminus was selectively more affected than its rod domain. Twenty-one days after injury, contractile properties were normal, fibers did not contain EBD, and dystrophin organization and protein level returned to normal. These data indicate the selective vulnerability of dystrophin after a single eccentric contraction-induced injury and suggest a critical role of dystrophin in force transduction.
Rapid muscle-specific gene expression changes after a single bout of eccentric contractions in the mouse
Authors: Barash Ilona A. , Mathew Liby , Ryan Allen F. , Chen Ju , Lieber Richard L.
Published: January 2004

Eccentric contractions (ECs), in which a muscle is forced to lengthen while activated, result in muscle injury and, eventually, muscle strengthening and prevention of further injury. Although the mechanical basis of EC-induced injury has been studied in detail, the biological response of muscle is less well characterized. This study presents the development of a minimally invasive model of EC injury in the mouse, follows the time course of torque recovery after an injurious bout of ECs, and uses Affymetrix microarrays to compare the gene expression profile 48 h after ECs to both isometrically stimulated muscles and contralateral muscles. Torque dropped by ∼55% immediately after the exercise bout and recovered to initial levels 7 days later. Thirty-six known genes were upregulated after ECs compared with contralateral and isometrically stimulated muscles, including five muscle-specific genes: muscle LIM protein (MLP), muscle ankyrin repeat proteins (MARP1 and -2; also known as cardiac ankyrin repeat protein and Arpp/Ankrd2, respectively), Xin, and myosin binding protein H. The time courses of MLP and MARP expression after the injury bout (determined by quantitative real-time polymerase chain reaction) indicate that these genes are rapidly induced, reaching a peak expression level of 6–11 times contralateral values 12–24 h after the EC bout and returning to baseline within 72 h. Very little gene induction was seen after either isometric activation or passive stretch, indicating that the MLP and MARP genes may play an important and specific role in the biological response of muscle to EC-induced injury.
The Ca2+-sensing receptor couples to Gα12/13 to activate phospholipase D in Madin-Darby canine kidney cells
Authors: Huang Chunfa , Hujer Kristine M. , Wu Zhenzhen , Miller R. Tyler
Published: January 2004

The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gαi to inhibit the activity of adenylyl cyclase and activate ERK, Gαq to stimulate phospholipase C and phospholipase A2, and Gβγ to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Gα12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Gα12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796W 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 CaRWT but not the CaRR796W. 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 Gα12/13. 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 Gαi or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gαi and Gαq, and a p115RhoGEF construct containing the RGS domain for Gα12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT 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 Gα12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of Gαi and Gαq. This suggests that the CaR may regulate cytoskeleton via Gα12/13, Rho, and PLD.
Effects of p38MAPK isoforms on renal mesangial cell inducible nitric oxide synthase expression
Authors: Lui Paul , Zeng Chenbo , Acton Stephen , Cok Steven , Sexton Alison , Morrison Aubrey R.
Published: January 2004

Several related isoforms of p38MAPK 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. p38α and -β2 are ubiquitously expressed, whereas p38γ and -δ 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 p38MAPK, with p38α mRNA expressed at the highest level, followed by p38γ and the lowest levels of expression by p38β2 and -δ. To determine the functional effects of these proteins on interleukin (IL)-1β-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 p38γ and -δ had minimal effects on iNOS expression, p38α and -β2 significantly altered its expression. p38α mutant and p38β2 wild-type dose dependently inhibited IL-1β-induced iNOS expression. These data suggest that p38α and β2 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 p38MAPK family of proteins.
Mitochondrial coupling in vivo in mouse skeletal muscle
Authors: Marcinek David J. , Schenkman Kenneth A. , Ciesielski Wayne A. , Conley Kevin E.
Published: January 2004

The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with 31P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 ± 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 ± 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought.
Rb regulates C/EBPβ-DNA-binding activity during 3T3-L1 adipogenesis
Authors: Cole Kathryn A. , Harmon Anne W. , Harp Joyce B. , Patel Yashomati M.
Published: January 2004

Two pathways are initiated upon 3T3-L1 preadipocyte differentiation: the reentry of cells into the cell cycle and the initiation of a cascade of transcriptional events that “prime” the cell for differentiation. The “priming” event involves the synthesis of members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. However, the relationship between these two pathways is unknown. Here we report that in the 3T3-L1 preadipocytes induced to differentiate, cell cycle progression and the initiation of differentiation are linked by a cell cycle-dependent Rb-C/EBPβ interaction. Cell cycle arrest in G1 by l-mimosine inhibited differentiation-induced C/EBPβ-DNA-binding activity and Rb phosphorylation. However, cell cycle arrest after the G1/S transition by aphidicolin or nocodazole did not prevent C/EBPβ-DNA-binding activity or Rb phosphorylation. Furthermore, hypophosphorylated Rb and C/EBPβ coimmunoprecipitated, whereas phosphorylated Rb and C/EBPβ did not. Electrophoretic mobility shift assays demonstrated that recombinant hypophosphorylated Rb decreased C/EBPβ-DNA-binding activity and that Rb overexpression inhibited C/EBPβ-induced transcriptional activation of a C/EBPα-promoter-luciferase reporter gene. We conclude that C/EBPβ-DNA-binding activity is regulated by its interaction with hypophosphorylated Rb, thereby linking the progression of the cell cycle to the initiation of differentiation during 3T3-L1 adipogenesis.
Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers
Authors: Henkin Josh A. , Maughan David W. , Vigoreaux Jim O.
Published: January 2004

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln0, a flightin null mutant, 2) Mhc13, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc6, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln0 and Mhc13 but not in Mhc6. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln0 and Mhc13 but not in Mhc6. Tension generation under isometric conditions was not impaired in fln0. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles.
An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell
Authors: McHugh J. M. , Kenyon J. L.
Published: January 2004

We wrote a program that runs as a Microsoft Excel spreadsheet to calculate the diffusion of Ca2+ in a spherical cell in the presence of a fixed Ca2+ buffer and two diffusible Ca2+ buffers, one of which is considered to be a fluorescent Ca2+ indicator. We modeled Ca2+ diffusion during and after Ca2+ influx across the plasma membrane with parameters chosen to approximate amphibian sympathetic neurons, mammalian adrenal chromaffin cells, and rat dorsal root ganglion neurons. In each of these cell types, the model predicts that spatially averaged intracellular Ca2+ activity ([Ca2+]avg) rises to a high peak and starts to decline promptly on the termination of Ca2+ influx. We compared [Ca2+]avg with predictions of ratiometric Ca2+ measurements analyzed in two ways. Method 1 sums the fluorescence at each of the two excitation or emission wavelengths over the N compartments of the model, calculates the ratio of the summed signals, and converts this ratio to Ca2+ ([Ca2+]avg,M1). Method 2 sums the measured number of moles of Ca2+ in each of the N compartments and divides by the volume of the cell ([Ca2+]avg,M2). [Ca2+]avg,M1 peaks well after the termination of Ca2+ influx at a value substantially less than [Ca2+]avg because the summed signals do not reflect the averaged free Ca2+ if the signals come from compartments containing gradients in free Ca2+ spanning nonlinear regions of the relationship between free Ca2+ and the fluorescence signals. In contrast, [Ca2+]avg,M2 follows [Ca2+]avg closely.
Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel
Authors: van Balkom Bas W. M. , Graat Michael P. J. , van Raak M. , Hofman Erik , van der Sluijs Peter , Deen Peter M. T.
Published: January 2004

In mammals, the regulation of water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which localizes to the basolateral and apical membranes of the early nephron segment, and AQP2, which is translocated from intracellular vesicles to the apical membrane of collecting duct cells after vasopressin stimulation. Because a similar localization and regulation are observed in transfected Madin-Darby Canine Kidney (MDCK) cells, we investigated which segments of AQP2 are important for its routing to forskolin-sensitive vesicles and the apical membrane through analysis of AQP1-AQP2 chimeras. AQP1 with the entire COOH tail of AQP2 was constitutively localized in the apical membrane, whereas chimeras with shorter COOH tail segments of AQP2 were localized in the apical and basolateral membrane. AQP1 with the NH2 tail of AQP2 was constitutively localized in both plasma membranes, whereas AQP1 with the NH2 and COOH tail of AQP2 was sorted to intracellular vesicles and translocated to the apical membrane with forskolin. These data indicate that region N220-S229 is essential for localization of AQP2 in the apical membrane and that the NH2 and COOH tail of AQP2 are essential for trafficking of AQP2 to intracellular vesicles and its shuttling to and from the apical membrane.
Activation of calcium release assessed by calcium release-induced inactivation of calcium current in rat cardiac myocytes
Authors: Zahradníková Alexandra , Kubalová Zuzana , Pavelková Jana , Györke Sándor , Zahradník Ivan
Published: January 2004

In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current ( ICa). We used this Ca2+ release-dependent inactivation (RDI) of ICa as a local probe of sarcoplasmic reticulum Ca2+ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca2+ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak ICa during a test pulse. The negative correlation between peak ICa suppression and ICa inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca2+ ions injected during the prepulse and during the subsequent tail current suppressed peak ICa in the test pulse to a different extent. Quantitative analysis indicated that equal Ca2+ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca2+ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca2+ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus.
Purification of interstitial cells of Cajal by fluorescence-activated cell sorting
Authors: Ördög Tamás , Redelman Doug , Miller Lisa J. , Horváth Viktor J. , Zhong Qiao , Almeida-Porada Graça , Zanjani Esmail D. , Horowitz Burton , Sanders Kenton M.
Published: January 2004

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000–40,000 cells and ∼60-fold enrichment of c- kit mRNA. However, the macrophage marker CD68 was also enriched ∼6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested ∼2,000–4,000 cells from single gastric corpus-antrum muscles and detected an ∼30-fold increase in c- kit mRNA, no enrichment of mast cells, and an ∼4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c- kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses.
Cruzipain, a major Trypanosoma cruzi antigen, promotes arginase-2 expression and survival of neonatal mouse cardiomyocytes
Authors: Aoki Maria P. , Guiñazú Natalia L. , Pellegrini Andrea V. , Gotoh Tomomi , Masih Diana T. , Gea Susana
Published: January 2004

An intense myocarditis is frequently found in the acute phase of Trypanosoma cruzi infection. Despite the cardiac damage, infected individuals may remain asymptomatic for decades. Thus T. cruzi may directly prevent cardiomyocyte death to keep heart destruction in check. Recently, it has been shown that Schwann cell invasion by T. cruzi, their prime target in the peripheral nervous system, suppressed host cell apoptosis caused by growth factor deprivation. Likewise, the trans-sialidase of T. cruzi reproduced this antiapoptotic activity of the parasite. In this study, we have investigated the effect of cruzipain, another important T. cruzi antigen, on survival and cell death of neonatal BALB/c mouse cardiomyocyte cultures. We have found that cruzipain, as well as T. cruzi infection, promoted survival of cardiomyocytes cultured under serum deprivation. The antiapoptotic effect was mediated by Bcl-2 expression but not by Bcl-xL expression. Because arginase activity is involved in cell differentiation and wound healing in most cell types and it favors parasite growth within the cell, we have further investigated the effect of cruzipain on the regulation of l-arginine metabolic pathways. Our results have revealed that cruzipain enhanced arginase activity and the expression of arginase-2 isoform but failed to induce nitric oxide synthase activity. In addition, the inhibition of arginase activity by NG-hydroxy-l-arginine, abrogated the antiapoptotic action of cruzipain. The results demonstrate that cruzipain may act as a survival factor for cardiomyocytes because it rescued them from apoptosis and stimulated arginase-2.
Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor
Authors: Meshki John , Tuluc Florin , Bredetean Ovidiu , Ding Zhongren , Kunapuli Satya P.
Published: January 2004

Nucleotides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y2 receptors. However, P2Y2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca2+ increases in isolated human neutrophils with an EC50 of 1 μM but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.
Copper-inducible transcription: regulation by metal- and oxidative stressresponsive pathways
Authors: Mattie Michael D. , Freedman Jonathan H.
Published: January 2004

Although copper is an essential metal, it is capable of catalyzing the formation of reactive oxygen species that can cause intracellular oxidative damage. We investigated the hypothesis that metal- and oxidative stress-responsive signal transduction pathways mediate the cellular and molecular responses associated with copper exposure. Transient transfection assays using COS-7 cells and mouse metallothionein-I (MT-I) or rat NAD(P)H:oxidoreductase 1-based reporter genes demonstrate that copper activates transcription via metal and antioxidant response elements. Concomitant with copper exposures is a decrease in the level of total glutathione and an increase in oxidized glutathione. Depletion of glutathione, before copper exposure, increases metal- and oxidative stress-inducible transcription and cytotoxicity. Pretreatment with the reactive oxygen scavengers aspirin or vitamin E provides partial protection against copper toxicity and reduces inducible transcription. Experiments using signal transduction inhibitors and a metal transcription factor (MTF)-1 null cell line demonstrate that copper-inducible MT-I transcription is regulated by protein kinase C and mitogen-activated protein kinase signaling pathways and requires MTF-1. The results of these studies indicate that copper activates transcription through both metal- and oxidative stress-responsive signal transduction pathways.
Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method
Authors: Solenov Eugen , Watanabe Hiroyuki , Manley Geoffrey T. , Verkman A. S.
Published: January 2004

A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible ∼12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 ± 0.05 s at 23°C, corresponding to an osmotic water permeability ( Pf) of ∼0.05 cm/s. Pf was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 ± 0.5 vs. 5.5 ± 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes.
Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1β
Authors: Kuramochi Yukio , Lim Chee Chew , Guo Xinxin , Colucci Wilson S. , Liao Ronglih , Sawyer Douglas B.
Published: January 2004

The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to β-adrenergic receptor (β-AR)-stimulated apoptosis, and neuregulin-1β (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to β-AR-stimulated JNK phosphorylation and cell death with 0.1 μM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 μM NE in quiescent cells. NRG suppressed β-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.
Cloning and characterization of mouse 5′-AMP-activated protein kinase γ3 subunit
Authors: Yu Haiyan , Fujii Nobuharu , Hirshman Michael F. , Pomerleau Jason M. , Goodyear Laurie J.
Published: January 2004

Naturally occurring mutations in the regulatory γ-subunit of 5′-AMP-activated protein kinase (AMPK) can result in pronounced pathological changes that may stem from increases in muscle glycogen levels, making it critical to understand the role(s) of the γ-subunit in AMPK function. In this study we cloned the mouse AMPKγ3 subunit and revealed that there are two transcription start sites, which result in a long form, γ3L (AF525500) and a short form, γ3S (AF525501). AMPKγ3L is the predominant form in mouse and is specifically expressed in mouse skeletal muscle at the protein level. In skeletal muscle, AMPKγ3 shows higher levels of expression in fast-twitch white glycolytic muscle (type IIb) compared with fast-twitch red oxidative glycolytic muscle (type IIa), whereas γ3 is undetectable in soleus muscle, a slow-twitch oxidative muscle with predominantly type I fibers. AMPKγ3 can coimmunoprecipititate with both α and β AMPK subunits. Overexpression of γ3S and γ3L in mouse tibialis anterior muscle in vivo has no effect on α1 and α2 subunit expression and does not alter AMPKα2 catalytic activity. However, γ3S and γ3L overexpression significantly increases AMPKα1 phosphorylation and activity by ∼50%. The increase in AMPKα1 activity is not associated with alterations in glycogen accumulation or glycogen synthase expression. In conclusion, the γ3 subunit of AMPK is highly expressed in fast-twitch glycolytic skeletal muscle, and wild-type γ3 functions in the regulation of α1 catalytic activity, but it is not associated with changes in muscle glycogen concentrations.
Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia
Authors: Kraft Robert , Grimm Christian , Grosse Karin , Hoffmann Anja , Sauerbruch Sophie , Kettenmann Helmut , Schultz Günter , Harteneck Christian
Published: January 2004

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H2O2), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H2O2 for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H2O2 and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H2O2 in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia.
Polymodal regulation of hTREK1 by pH, arachidonic acid, and hypoxia: physiological impact in acidosis and alkalosis
Authors: Miller Paula , Peers Chris , Kemp Paul J.
Published: January 2004

Expression of the human tandem P domain K+ channel, hTREK1, is limited almost exclusively to the central nervous system, where ambient Po2 can be as low as 20 Torr. We have previously shown that this level of hypoxia evokes a maximal inhibitory influence on recombinant hTREK1 and occludes the activation by arachidonic acid; this has cast doubt on the idea that TREK1 activation during brain ischemia could facilitate neuroprotection via hyperpolarizing neurons in which it is expressed. Using both whole cell and cell-attached patch-clamp configurations, we now show that the action of another potent TREK activator and ischemia-related event, intracellular acidification, is similarly without effect during compromised O2 availability. This occlusion is observed in either recording condition, and even the concerted actions of both arachidonic acid and intracellular acidosis are unable to activate hTREK1 during hypoxia. Conversely, intracellular alkalinization is a potent channel inhibitor, and hypoxia does not reverse this inhibition. However, increases in intracellular pH are unable to occlude either arachidonic acid activation or hypoxic inhibition. These data highlight two important points. First, during hypoxia, modulation of hTREK1 cannot be accomplished by parameters known to be perturbed in brain ischemia (increased extracellular fatty acids and intracellular acidification). Second, the mechanism of regulation by intracellular alkalinization is distinct from the overlapping structural requirements known to exist for regulation by arachidonic acid, membrane distortion, and acidosis. Thus it seems likely that hTREK1 regulation in the brain will be physiologically more relevant during alkalosis than during ischemia or acidosis.
Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands
Authors: Venkatesh S. G. , Cowley Darrin J , Gorr Sven-Ulrik
Published: January 2004

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.
Involvement of PKCδ and PKD in pulmonary microvascular endothelial cell hyperpermeability
Authors: Tinsley John H. , Teasdale Nicole R. , Yuan Sarah Y.
Published: January 2004

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (α, βI, βII, and γ), novel [δ, ϵ, η, and μ (also known as PKD),θ], and atypical (ζ and ι/λ), 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 α, βI, and ϵ and complete translocation of PKCδ and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKCδ and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms α, βI, and ϵ were dispensable with regard to these same phenomena.
Serine protease activation of near-silent epithelial Na+ channels
Authors: Caldwell Ray A. , Boucher Richard C. , Stutts M. Jackson
Published: January 2004

The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability ( Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat α-, β-, and γ-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity ( NPo) < 0.03, where N is the number of channels. Within 1–5 min of 3 μg/ml bath trypsin superfusion, NPo increased ∼66-fold ( n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 ± 0.01 to 0.57 ± 0.03 ( n = 3, mean ± SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels.
[Ca2+]i regulates trafficking of Cav1.3 (α1D Ca2+ channel) in insulin-secreting cells
Authors: Huang Luping , Bhattacharjee Arin , Taylor James T. , Zhang Min , Keyser Brian M. , Marrero Luis , Li Ming
Published: January 2004

Chronic exposure of pancreatic β-cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of β-cell basal intracellular free Ca2+ concentration ([Ca2+]i). We have investigated the relationship between the basal intracellular free Ca2+ and the L-type (Cav1.3) Ca2+ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca2+ influx, which was attenuated when the basal [Ca2+]i was elevated. Using voltage-clamp techniques, we found that changing [Ca2+]i could regulate the amplitude of the Ca2+ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Cav1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca2+]i conditions by deconvolution microscopy. The [Ca2+]i-dependent translocation of Cav1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca2+]i. These results suggest that Cav1.3 channel trafficking is involved in glucose desensitization of pancreatic β-cells.
TRPV4 calcium entry channel: a paradigm for gating diversity
Authors: Nilius Bernd , Vriens Joris , Prenen Jean , Droogmans Guy , Voets Thomas
Published: January 2004

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca2+ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4α-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of Ca2+ entry channels.