bluuebunny/crossref_metadata_2025 huggingface dataset:
α1C (CaV1.2) L-type calcium channel mediates mechanosensitive calcium regulation
Authors: Lyford Greg L. , Strege Peter R. , Shepard Allan , Ou Yijun , Ermilov Leonid , Miller Steven M. , Gibbons Simon J. , Rae James L. , Szurszewski Joseph H. , Farrugia Gianrico
Published: May 2013

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the α1C L-type calcium channel subunit (CaV1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a β2 calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac α1C splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the α1C COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming α1C-subunit.
Modulation of the erythropoietin-induced proliferative pathway by cAMP in vascular smooth muscle cells
Authors: Ito Chiharu , Kusano Eiji , Furukawa Yusuke , Yamamoto Hisashi , Takeda Shin-Ichi , Akimoto Tetsu , Iimura Osamu , Ando Yasuhiro , Asano Yasushi
Published: May 2013

We previously reported that erythropoietin (Epo) has a mitogenic effect on rat vascular smooth muscle cells (VSMC) and that activation of the mitogen-activated protein kinase (MAPK) cascade is an important mediator for Epo-induced mitogenesis. An increase in intracellular cAMP has an antiproliferative effect on VSMC. We therefore hypothesized that cAMP effectors inhibit Epo-induced MAPK activation in rat VSMC. When we exposed VSMC to recombinant human Epo (rHuEpo), DNA synthesis was increased. Forskolin (Fsk) or cilostazol (Cil) decreased the DNA synthesis stimulated by rHuEpo. Coincubation with Rp-cAMPS triethylamine canceled the suppression of DNA synthesis and MAPK activity by Fsk. Both rHuEpo and phorbol 12-myristate 13-acetate upregulated phosphorylations of MEK and MAPK. Pretreatment with Fsk inhibited these phosphorylations. Protein kinase C inhibitors also suppressed MEK and MAPK phosphorylations. Moreover, Fsk induced phosphorylation of Raf-1 at serine-259. These results indicated that cAMP inhibited Epo-induced MAPK activation and that this suppression might be regulated upstream or at Raf-1. The results also suggested that these agents, which could accumulate cAMP, might be protective for Epo-stimulated direct action.
Low-voltage triggering of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle cells
Authors: Brette Fabien , Le Guennec Jean-Yves , Findlay Ian
Published: May 2013

This study investigated the interaction between L-type Ca2+ current (ICaL) and Ca2+ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Nao+:KI+) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca2+ current, TTX-sensitive ICa (ICaTTX), or “slip mode” Ca2+ conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca2+ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca2+ release from the SR at negative potentials is due to activation of L-type Ca2+ channels.
Cu2+-induced modification of the kinetics of Aβ(1-42) channels
Authors: Bahadi Randa , Farrelly Peter V. , Kenna Bronwyn L. , Curtain Cyril C. , Masters Colin L. , Cappai Roberto , Barnham Kevin J. , Kourie Joseph I.
Published: May 2013

We found that the amyloid β peptide Aβ(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu2+ or biological redox agents that have been reported to mediate Aβ(1-42) toxicity. The Aβ(1-42)-formed cation channel was inhibited by Cu2+ in cis solution ([Cu2+] cis) in a voltage- and concentration-dependent manner between 0 and 250 μM. The [Cu2+] cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 μM [Cu2+] cis and partially reversible at 250 μM [Cu2+] cis. The inhibitory effects of [Cu2+] cis between 50 and 250 μM on the channel could not be reversed with addition of Cu2+-chelating agent clioquinol (CQ) at concentrations between 64 and 384 μM applied to the cis chamber. The effects of 200-250 μM [Cu2+] cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 μM [CQ] cis. The kinetic analysis of the data indicate that Cu2+-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu2+ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu2+-binding site of the Aβ(1-42)-formed channels is modulated with Cu2+ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu2+ modulation of Aβ and PrP channel proteins linked to neurodegenerative diseases.
Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells
Authors: Leung Lawrence W. , Contreras Ruben G. , Flores-Maldonado Catalina , Cereijido Marcelino , Rodriguez-Boulan Enrique
Published: May 2013

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Ω · cm2. When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 Ω · cm2. These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.
M20, the small subunit of PP1M, binds to microtubules
Authors: Takizawa Norio , Schmidt David J. , Mabuchi Katsuhide , Villa-Moruzzi Emma , Tuft Richard A. , Ikebe Mitsuo
Published: May 2013

Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics.
β1-Subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C
Authors: Hagen Brian M. , Bayguinov Orline , Sanders Kenton M.
Published: May 2013

Colonic myocytes have spontaneous, localized, Ins ( 1 , 4 , 5 ) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory β1-subunit of BK channels. Myocytes from β1-knockout (β1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of β1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between α- and β1-subunits.
Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity
Authors: Li Hongyan , Pajor Ana M.
Published: May 2013

The human Na+-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na+-sulfate cotransporter (hSUT-1) and the Na+-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide- N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn591 is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced Vmax, with no change in Km. These results suggest that Asn591 and/or N-glycosylation is critical for transport activity in NaSi-1.
Role for phosphoinositide 3-kinase in FcγRIIA-induced platelet shape change
Authors: Barkalow Kurt L. , Falet Hervé , Italiano Joseph E. , van Vugt Andrew , Carpenter Christopher L. , Schreiber Alan D. , Hartwig John H.
Published: May 2013

Platelets transform from disks to irregular spheres, grow filopodia, form ruffles, and spread on surfaces coated with anti-FcγRIIA antibody. FcγRIIA cross-linking leads to a tenfold increase in actin filament barbed end exposure and robust actin assembly. Activation of the small GTPases Rac and Cdc42 follows FcγRIIA cross-linking. Shape change, actin filament barbed end exposure, and quantifiable actin assembly require phosphoinositide 3-kinase (PI3-kinase) activity and a rise in intracellular calcium. PI3-kinase inhibition blocks activation of Rac, but not of Cdc42, and diminishes the association of Arp2/3 complex and CapZ with polymerized actin. Furthermore, addition of constitutively active D-3 phosphorylated polyphosphoinositides or recombinant PI3-kinase subunits to octylglucoside-permeabilized platelets elicits actin filament barbed end exposure by releasing gelsolin and CapZ from the cytoskeleton. Our findings place PI3-kinase activity upstream of Rac, gelsolin, and Arp2/3 complex activation induced by FcγRIIA and clearly distinguish the FcγRIIA signaling pathway to actin filament assembly from the thrombin receptor protease-activated receptor (PAR)-1 pathway.
Cellular and molecular responses to increased skeletal muscle loading after irradiation
Authors: Adams Gregory R. , Caiozzo Vincent J. , Haddad Fadia , Baldwin Kenneth M.
Published: May 2013

Irradiation of rat skeletal muscles before increased loading has been shown to prevent compensatory hypertrophy for periods of up to 4 wk, possibly by preventing satellite cells from proliferating and providing new myonuclei. Recent work suggested that stem cell populations exist that might allow irradiated muscles to eventually hypertrophy over time. We report that irradiation essentially prevented hypertrophy in rat muscles subjected to 3 mo of functional overload (OL-Ir). The time course and magnitude of changes in cellular and molecular markers of anabolic and myogenic responses were similar in the OL-Ir and the contralateral nonirradiated, overloaded (OL) muscles for the first 3–7 days. These markers then returned to control levels in OL-Ir muscles while remaining elevated in OL muscles. The number of myonuclei and amount of DNA were increased markedly in OL but not OL-Ir muscles. Thus it appears that stem cells were not added to the irradiated muscles in this time period. These data are consistent with the theory that the addition of new myonuclei may be required for compensatory hypertrophy in the rat.
Regulation of elastin gene transcription by interleukin-1β-induced C/EBPβ isoforms
Authors: Kuang Ping-Ping , Goldstein Ronald H.
Published: May 2013

We previously showed that interleukin (IL)-1β decreases elastin gene transcription through activation of the NF-κB subunit p65 in neonatal rat lung fibroblasts. The present study was undertaken to further explore the molecular mechanisms responsible for the inhibitory effect of IL-1β on elastin gene transcription. We found that cycloheximide blocked IL-1β-induced downregulation of elastin mRNA but did not inhibit IL-1β-induced translocation of p65 into the nucleus. IL-1β treatment increased CCAAT/enhancer-binding protein (C/EBP)β mRNA and protein levels including liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP), which was cycloheximide sensitive. C/EBPβ isoforms bound a GCAAT-containing sequence in the proximal elastin promoter as determined by electrophoretic gel shift studies and confirmed by using specific anti-C/EBPβ antibodies and by competition studies with oligonucleotides. Transient transfection of LIP expression vectors strongly decreased the transcriptional activity of the cotransfected elastin promoter and decreased levels of endogenous elastin mRNA. We demonstrated that IL-1β-induced downregulation of elastin mRNA is dependent on NF-κB activation and C/EBPβ expression. These results indicate that IL-1β treatment activates NF-κB, which subsequently induces LIP expression and inhibition of elastin gene transcription.
ENaC plays a role in regulated antibody secretion by hybridomas
Authors: Zhou Zhen-Hong , Bubien James K.
Published: May 2013

Hybridomas are fused immortal lymphocytes that typically secrete monoclonal antibodies to a known antigen. Hybridomas express two ionic conductances that have properties consistent with epithelial sodium channel (ENaC) and CFTR. Both ion channels are expressed by lymphocytes. Both of these channels are known to play a role in epithelial cell physiology. However, the physiological role of these channels in lymphocytes is unclear. We tested the hypothesis that ENaC plays a role in the process of regulated antibody secretion. We have been able to demonstrate that hybridomas can be provoked to acutely secrete monoclonal antibodies by a variety of agonists. Concurrently, we were able to show that these same agonists activate amiloride-sensitive sodium currents in whole cell clamped hybridomas. Inhibition of ENaC by amiloride inhibited the acute provoked antibody secretion, thereby linking ENaC to the process of acute antibody secretion. Interestingly, the concentration of amiloride necessary to completely inhibit the provoked secretion was approximately an order of magnitude higher than the concentration necessary to inhibit all of the transmembrane current. However, because amiloride is a weak base, the equilibrium concentration necessary to produce partial inhibition was precisely in accord with the K i for amiloride and ENaC, indicating that the inhibition was intracellular.
Regulation of Ca2+ release-activated Ca2+channels by INAD and Ca2+ influx factor
Authors: Su Zhengchang , Barker Douglas S. , Csutora Peter , Chang Theresa , Shoemaker Richard L. , Marchase Richard B. , Blalock J. Edwin
Published: May 2013

The coupling mechanism between depletion of Ca2+ stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to Ca2+ signaling in many cell types and has yet to be completely elucidated. Using Ca2+release-activated Ca2+ (CRAC) channels in RBL-2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires Ca2+ and can be reversed by a diffusible Ca2+ influx factor. Thus the coupling between the depletion of Ca2+ stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low-molecular-weight, diffusible store-depletion signal.
Effect of Pi on unloaded shortening velocity of slow and fast mammalian muscle fibers
Authors: Widrick Jeffrey J.
Published: May 2013

Chemically skinned muscle fibers, prepared from the rat medial gastrocnemius and soleus, were subjected to four sequential slack tests in Ca2+-activating solutions containing 0, 15, 30, and 0 mM added Pi. Pi (15 and 30 mM) had no effect on the unloaded shortening velocity ( V o) of fibers expressing type IIb myosin heavy chain (MHC). For fibers expressing type I MHC, 15 mM Pi did not alter V o, whereas 30 mM Pireduced V o to 81 ± 1% of the original 0 mM Pi value. This effect was readily reversible when Pi was lowered back to 0 mM. These results are not compatible with current cross-bridge models, developed exclusively from data obtained from fast fibers, in which V o is independent of Pi. The response of the type I fibers at 30 mM Pi is most likely the result of increased internal drag opposing fiber shortening resulting from fiber type-specific effects of Pi on cross bridges, the thin filament, or the rate-limiting step of the cross-bridge cycle.
Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gβγ-subunits in Balb/c-3T3 cells
Authors: Liu Gele , Ghahremani Mohammad H. , Banihashemi Behzad , Albert Paul R.
Published: May 2013

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC50 = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating Gi/Go proteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Gαi proteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Gαi2 nor Gαi3 rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gβγ-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17β]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gβγ-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.
Glutamate 172, essential for modulation of L247T α7 ACh receptors by Ca2+, lines the extracellular vestibule
Authors: Eddins Donnie , Sproul Adrian D. , Lyford Lisa K. , McLaughlin James T. , Rosenberg Robert L.
Published: May 2013

Neuronal α7 nicotinic ACh receptors (nAChRs) are permeable to and modulated by Ca2+, Ba2+, and Sr2+. These permeant divalent cations interact with slowly desensitizing L247T α7nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro-β-erythroidine (DHβE), and increase agonist-independent activity. Mutation of glutamate 172 (E172) to glutamine or cysteine eliminated these effects of permeant divalent cations. 2-(Trimethylammonium)ethyl methanethiosulfonate (MTSET), a cysteine-modifying reagent directed at water-accessible thiols, inhibited ACh-evoked currents of E172C/L247T α7 nAChRs by >90%, demonstrating that E172 was accessible to permeant ions. The data are consistent with a model of α7receptors, derived from the crystal structure of the ACh binding protein (AChBP) from Lymnaea stagnalis, in which E172 projects toward the lumen of the extracellular vestibule. The observations that E172 was essential for divalent cation modulation of L247T α7 nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity.
Differential roles of ICAM-1 and E-selectin in polymorphonuclear leukocyte-induced angiogenesis
Authors: Yasuda Masako , Shimizu Shunichi , Ohhinata Kyoko , Naito Shinji , Tokuyama Shogo , Mori Yasuo , Kiuchi Yuji , Yamamoto Toshinori
Published: May 2013

Ets-1, which stimulates metalloproteinase gene transcription, has a key role in angiogenesis. We first examined whether activated polymorphonuclear leukocytes (PMNs) enhanced angiogenesis through the induction of Ets-1. Addition of activated PMNs to endothelial cells stimulated both in vitro angiogenesis in collagen gel and Ets-1 expression. Both angiogenesis and Ets-1 expression induced by PMNs were reduced by ets-1 antisense oligonucleotide, suggesting that Ets-1 is an important factor in PMN-induced angiogenesis. Although intercellular adhesion molecule (ICAM)-1 and E-selectin are involved in PMN-induced angiogenesis, the mechanisms underlying their roles in angiogenesis have yet to be elucidated. PMN-induced Ets-1 expression was reduced by a monoclonal antibody against ICAM-1 but not E-selectin despite the inhibition of PMN-induced angiogenesis by both antibodies. Moreover, the stimulation of angiogenesis by H2O2 without PMNs was inhibited by a monoclonal antibody to E-selectin but not ICAM-1. These findings suggested that ICAM-1 in endothelial cells may act as a signaling receptor to induce Ets-1 expression, whereas E-selectin seems to function in the formation of tubelike structures in vascular endothelial cell cultures.
Dual effect of nitric oxide on cytosolic Ca2+concentration and insulin secretion in rat pancreatic β-cells
Authors: Kaneko Yukiko , Ishikawa Tomohisa , Amano Satoshi , Nakayama Koichi
Published: May 2013

In isolated rat pancreatic β-cells, the nitric oxide (NO) donor NOC-7 at 1 μM reduced the amplitude of the oscillations of cytosolic Ca2+ concentration ([Ca2+]c) induced by 11.1 mM glucose, and at 10 μM terminated them. In the presence of N G-nitro-l-arginine (l-NNA), however, NOC-7 at 0.5 and 1 μM increased the amplitude of the [Ca2+]c oscillations, although the NO donor at 10 μM still suppressed them. Aqueous NO solution also had a dual effect on the [Ca2+]c oscillations. The soluble guanylate cyclase inhibitor LY-83583 and the cGMP-dependent protein kinase inhibitor KT5823 inhibited the stimulatory effect of NO, and 8-bromo-cGMP increased the amplitude of the [Ca2+]c oscillations. Patch-clamp analyses in the perforated configuration showed that 8-bromo-cGMP inhibited whole cell ATP-sensitive K+ currents in the isolated rat pancreatic β-cells, suggesting that the inhibition by cGMP of ATP-sensitive K+ channels is, at least in part, responsible for the stimulatory effect of NO on the [Ca2+]c oscillations. In the presence ofl-NNA, the glucose-induced insulin secretion from isolated islets was facilitated by 0.5 μM NOC-7, whereas it was suppressed by 10 μM NOC-7. These results suggest that NO facilitates glucose-induced [Ca2+]c oscillations of β-cells and insulin secretion at low concentrations, which effects are mediated by cGMP, whereas NO inhibits them in a cGMP-independent manner at high concentrations.
C2C12 myoblast/osteoblast transdifferentiation steps enhanced by epigenetic inhibition of BMP2 endocytosis
Authors: Rauch Cyril , Brunet Anne-Christine , Deleule Julie , Farge Emmanuel
Published: May 2013

We investigated the modulation of critical transcriptional steps of C2C12myoblast/osteoblast transdifferentiation triggered by the bone morphogenetic protein 2 (BMP2) signaling protein, in response to epigenetic inhibition of the endocytotic internalization of exogenous BMP2. BMP2 endocytosis was inhibited chemically with polyethylene glycol-50 (PEG-Chol) and cyclodextrin and mechanically by mild hyposmotic treatment. BMP2-dependent nuclear translocation of the mother against Dpp (Smad1) transcription factor was ten times faster if BMP2 endocytosis was inhibited. Smad1-dependent expression of the JunB gene, the first transcriptional step in myoblast dedifferentiation, was increased by a factor of three to four. JunB-dependent levels of myogenin repression, one of the critical markers of terminal myoblastic differentiation, was amplified by a factor of three. Smad1-dependent levels of alkaline phosphatase expression, one of the C2C12 osteoblast differentiation markers, were 3.5 to 5 times higher. The same behavior was observed for osteopontin, the other C2C12 osteoblast differentiation marker. These results suggest that the cell genome could “sense” tissue mechanical deformations by mechanical inhibition of signaling protein endocytosis, thereby translating mechanical strains into transcription events involved in cell differentiation.
Elevated L-PGDS activity contributes to PMA-induced apoptosis concomitant with downregulation of PI3-K
Authors: Ragolia Louis , Palaia Thomas , Paric Enesa , Maesaka John K.
Published: May 2013

Recently we demonstrated the induction of apoptosis by the addition of recombinant lipocalin-type prostaglandin D2 synthase (L-PGDS) to the culture medium of LLC-PK1 cells. Because protein kinase C (PKC) has been shown to be involved in the apoptotic process of various cell types, we examined the potential role of L-PGDS in phorbol 12-myristate 13-acetate (PMA)-induced apoptosis. We report here the enzymatic activation and phosphorylation of L-PGDS in response to phorbol ester in cell culture and the direct phosphorylation of recombinant L-PGDS by PKC in vitro. Treatment of cells with PMA or L-PGDS decreased phosphatidylinositol 3-kinase (PI3-K) activity and concomitantly inhibited protein kinase B (PKB/Akt) phosphorylation, which led to the hypophosphorylation and activation of Bad. In addition, hypophosphorylation of retinoblastoma protein was also observed in response to L-PGDS-induced apoptosis. Cellular depletion of L-PGDS levels by using an antisense RNA strategy prevented PI3-K inactivation by phorbol ester and inhibited caspase-3 activation and apoptosis. We conclude that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by PKC and is accompanied by inhibition of the PI3-K/PKB anti-apoptotic signaling pathways.
Regulation of endothelial nitric oxide synthase by the actin cytoskeleton
Authors: Su Yunchao , Edwards-Bennett Sophia , Bubb Michael R. , Block Edward R.
Published: May 2013

In the present study, the association of endothelial nitric oxide synthase (eNOS) with the actin cytoskeleton in pulmonary artery endothelial cells (PAEC) was examined. We found that the protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAEC resulted in the coprecipitation of actin, and immunoprecipitation of actin from lysates of caveolar fractions resulted in the coprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS was labeled with fluorescein, F-actin was labeled with Texas red-phalloidin, and G-actin was labeled with deoxyribonuclease I conjugated with Texas red, also demonstrated an association between eNOS and F-actin or G-actin. Incubation of purified eNOS with purified F-actin and G-actin resulted in an increase in eNOS activity. The increase in eNOS activity caused by G-actin was much higher than that caused by F-actin. Incubation of PAEC with swinholide A, an actin filament disruptor, resulted in an increase in eNOS activity, eNOS protein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activity was higher than that in eNOS protein content in swinholide A-treated cells. In contrast, exposure of PAEC to phalloidin, an actin filament stabilizer, caused decreases in eNOS activity and association of eNOS with G-actin and increases in association of eNOS with F-actin. These results suggest that eNOS is associated with actin in PAEC and that actin and its polymerization state play an important role in the regulation of eNOS activity.
Mechanism linking NMDA receptor activation to modulation of voltage-gated sodium current in distal retina
Authors: Davis Scott F. , Linn Cindy L.
Published: May 2013

In this study, we investigated the mechanism that links activation of N-methyl-D-aspartate (NMDA) receptors to inhibition of voltage-gated sodium channels in isolated catfish cone horizontal cells. NMDA channels were activated in voltage-clamped cells incubated in low-calcium saline or dialyzed with the calcium chelator BAPTA to determine that calcium influx through NMDA channels is required for sodium channel modulation. To determine whether calcium influx through NMDA channels triggers calcium-induced calcium release (CICR), cells were loaded with the calcium-sensitive dye calcium green 2 and changes in relative fluorescence were measured in response to NMDA. Responses were compared with measurements obtained when caffeine depleted stores. Voltage-clamp studies demonstrated that CICR modulated sodium channels in a manner similar to that of NMDA. Blocking NMDA receptors with AP-7, blocking CICR with ruthenium red, depleting stores with caffeine, or dialyzing cells with calmodulin antagonists W-5 or peptide 290–309 all prevented sodium channel modulation. These results support the hypothesis that NMDA modulation of voltage-gated sodium channels in horizontal cells requires CICR and activation of a calmodulin-dependent signaling pathway.
Parathyroid hormone-related protein ameliorates death receptor-mediated apoptosis in lung cancer cells
Authors: Hastings Randolph H. , Araiza Flavio , Burton Douglas W. , Zhang Lu , Bedley Maxwell , Deftos Leonard J.
Published: May 2013

Parathyroid hormone-related protein (PTHrP) is expressed in more advanced, aggressive tumors and may play an active role in cancer progression. This study investigated the effects of PTHrP on apoptosis after UV irradiation, Fas ligation, or staurosporine treatment in BEN human squamous lung carcinoma cells. Cells at 70% confluency were treated for 24 h with 100 nM PTHrP-(1-34), PTHrP-(38-64), PTHrP-(67-86), PTHrP-(107-139), or PTHrP-(140-173) in media with serum, exposed for 30 min to UV-B radiation (0.9 mJ/cm2), and maintained for another 24 h. Caspase-3, caspase-8, and caspase-9 activities increased fivefold. Pretreatment with PTHrP-(1-34) and PTHrP-(140-173) ameliorated apoptosis after UV irradiation, as indicated by reduced caspase activities, increased cell protein, decreased nuclear condensation, and increased clonal survival. Other peptides had no effect on measures of apoptosis. PTHrP-(140-173) also reduced caspase activities after Fas ligation by activating antibody, but neither peptide had effects on caspase-3 or caspase-9 activity after 1 μM staurosporine. These data indicate that PTHrP-(1-34) and PTHrP-(140-173) protect against death receptor-induced apoptosis in BEN lung cancer cells but are ineffective against mitochondrial pathways. PTHrP contributes to lung cancer cell survival in culture and could promote cancer progression in vivo. The mechanism for the protective effect against apoptosis remains to be determined.
Cell prestress. II. Contribution of microtubules
Authors: Stamenović Dimitrije , Mijailovich Srboljub M. , Tolić-Nørrelykke Iva Marija , Chen Jianxin , Wang Ning
Published: May 2013

The tensegrity model hypothesizes that cytoskeleton-based microtubules (MTs) carry compression as they balance a portion of cell contractile stress. To test this hypothesis, we used traction force microscopy to measure traction at the interface of adhering human airway smooth muscle cells and a flexible polyacrylamide gel substrate. The prediction is that if MTs balance a portion of contractile stress, then, upon their disruption, the portion of stress balanced by MTs would shift to the substrate, thereby causing an increase in traction. Measurements were done first in maximally activated cells (10 μM histamine) and then again after MTs had been disrupted (1 μM colchicine). We found that after disruption of MTs, traction increased on average by ∼13%. Because in activated cells colchicine induced neither an increase in intracellular Ca2+ nor an increase in myosin light chain phosphorylation as shown previously, we concluded that the observed increase in traction was a result of load shift from MTs to the substrate. In addition, energy stored in the flexible substrate was calculated as work done by traction on the deformation of the substrate. This result was then utilized in an energetic analysis. We assumed that cytoskeleton-based MTs are slender elastic rods supported laterally by intermediate filaments and that MTs buckle as the cell contracts. Using the post-buckling equilibrium theory of Euler struts, we found that energy stored during buckling of MTs was quantitatively consistent with the measured increase in substrate energy after disruption of MTs. This is further evidence supporting the idea that MTs are intracellular compression-bearing elements.
Chloride secretion by semicircular canal duct epithelium is stimulated via β2-adrenergic receptors
Authors: Milhaud Pierre G. , Pondugula Satyanarayana R. , Lee Jun Ho , Herzog Michael , Lehouelleur Jacques , Wangemann Philine , Sans Alain , Marcus Daniel C.
Published: May 2013

The ductal epithelium of the semicircular canal forms much of the boundary between the K+-rich luminal fluid and the Na+-rich abluminal fluid. We sought to determine whether the net ion flux producing the apical-to-basal short-circuit current ( I sc) in primary cultures was due to anion secretion and/or cation absorption and under control of receptor agonists. Net fluxes of 22Na, 86Rb, and36Cl demonstrated a basal-to-apical Cl−secretion that was stimulated by isoproterenol. Isoproterenol and norepinephrine increased I sc with an EC50 of 3 and 15 nM, respectively, and isoproterenol increased tissue cAMP of native canals with an EC50 of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had no effect on I sc. Isoproterenol stimulation of I sc and cAMP was inhibited by ICI-118551 (IC50 = 6 μM for I sc) but not by CGP-20712A (1 μM) in primary cultures, and similar results were found in native epithelium. I sc was partially inhibited by basolateral Ba2+ (IC50 = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, and DIDS did not fully fit the profile for CFTR. Our findings show that the canal epithelium contributes to endolymph homeostasis by secretion of Cl− under β2-adrenergic control with cAMP as second messenger, a process that parallels the adrenergic control of K+ secretion by vestibular dark cells. The current work points to one possible etiology of endolymphatic hydrops in Meniere's disease and may provide a basis for intervention.
Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl– channels in human osteoblasts
Authors: Bräuer Margot , Frei Eva , Claes Lutz , Grissmer Stephan , Jäger Heike
Published: May 2013

The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl– channels expressed in these cells as described previously. The reversal potential of the induced Cl– current is more negative when activated through hypotonic NaCl solution (–47 ± 5 mV; n = 6) compared with activation through isotonic high-KCl solution (–35 ± 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl–] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl– current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1–KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl– channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling.
Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers
Authors: Karatzaferi Christina , Myburgh Kathryn H. , Chinn Marc K. , Franks-Skiba Kathleen , Cooke Roger
Published: May 2013

The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1–2 mM, and [ATP], 0.05–0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P′), the absolute value of P′ being nearly constant, ∼0.04 N/mm2, in variable ATP levels, pH 7. Tension decreased (50–60%) at pH 6, but upon addition of SL-ADP, P′ was still ∼0.04 N/mm2. The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K i, of ∼240 and 280 μM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.
Effect of endothelial cell polarity on β-amyloid-induced migration of monocytes across normal and AD endothelium
Authors: Giri Ranjit , Selvaraj Suresh , Miller Carol A. , Hofman Florence , Yan S. D. , Stern David , Zlokovic Berislav V. , Kalra Vijay K.
Published: May 2013

During normal aging and amyloid β-peptide (Aβ) disorders such as Alzheimer's disease (AD), one finds increased deposition of Aβ and activated monocytes/microglial cells in the brain. Our previous studies show that Aβ interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Aβ accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Aβ1—40 with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Aβ was added to the basolateral side of AD compared with normal individual BEC. The Aβ-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Aβ receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Aβ with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Aβ in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.
A-type potassium currents in smooth muscle
Authors: Amberg Gregory C. , Koh Sang Don , Imaizumi Yuji , Ohya Susumu , Sanders Kenton M.
Published: May 2013

A-type currents are voltage-gated, calcium-independent potassium (Kv) currents that undergo rapid activation and inactivation. Commonly associated with neuronal and cardiac cell-types, A-type currents have also been identified and characterized in vascular, genitourinary, and gastrointestinal smooth muscle cells. This review examines the molecular identity, biophysical properties, pharmacology, regulation, and physiological function of smooth muscle A-type currents. In general, this review is intended to facilitate the comparison of A-type currents present in different smooth muscles by providing a comprehensive report of the literature to date. This approach should also aid in the identification of areas of research requiring further attention.
Characterization of l-glutamine transport by a human neuroblastoma cell line
Authors: Wasa Masafumi , Wang Hong-Sheng , Okada Akira
Published: May 2013

This study characterized the Na+-dependent transport of l-glutamine by a human neuroblastoma cell line, SK-N-SH. The Na+-dependent component represented >95% of the total glutamine uptake. Kinetic studies showed a single saturable high-affinity carrier with a Michaelis constant ( K m) of 163 ± 23 μM and a maximum transport velocity ( V max) of 13,713 ± 803 pmol · mg protein−1 · min−1. Glutamine uptake was markedly inhibited in the presence of l-alanine,l-asparagine, and l-serine. Li+ did not substitute for Na+. These data show thatl-glutamine is predominantly taken up through system ASC. Glutamine deprivation resulted in the decrease of glutamine transport by a mechanism that decreased V max without affecting K m. The expression of the system ASC subtype ASCT2 decreased in the glutamine-deprived group, whereas glutamine deprivation did not induce changes in system ASC subtype ASCT1 mRNA expression. Adaptive increases in Na+-dependent glutamate, Na+-dependent 2-(methylamino)isobutyric acid, and Na+-independent leucine transport were observed under glutamine-deprived conditions, which were completely blocked by actinomycin D and cycloheximide. These mechanisms may allow cells to survive and even grow under nutrient-deprived conditions.
Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production
Authors: Sayen M. R. , Gustafsson Åsa B. , Sussman Mark A. , Molkentin Jeffery D. , Gottlieb Roberta A.
Published: May 2013

Introduction of the constitutively active calcineurin gene into neonatal rat cardiomyocytes by adenovirus resulted in decreased mitochondrial membrane potential ( P < 0.05). Infection of H9c2 cells with calcineurin adenovirus resulted in increased superoxide production ( P < 0.001). Transgenic mice with cardiac-specific expression of a constitutively active calcineurin cDNA (CalTG mice) exhibit a two- to threefold increase in heart size that progresses to heart failure. We prepared mitochondria enriched for the subsarcolemmal population from the hearts of CalTG mice and transgene negative littermates (control). Intact, well-coupled mitochondria prepared from one to two mouse hearts at a time yielded sufficient material for functional studies. Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode with substrates for mitochondrial complex II (succinate) and complex IV [tetramethylpentadecane (TMPD)/ascorbate]. CalTG mice exhibited a maximal rate of electron transfer in heart mitochondria that was reduced by ∼50% ( P < 0.002) without a loss of respiratory control. Mitochondrial respiration was unaffected in tropomodulin-overexpressing transgenic mice, another model of cardiomyopathy. Western blotting for mitochondrial electron transfer subunits from mitochondria of CalTG mice revealed a 20–30% reduction in subunit 3 of complex I (ND3) and subunits I and IV of cytochrome oxidase (CO-I, CO-IV) when normalized to total mitochondrial protein or to the adenine nucleotide transporter (ANT) and compared with littermate controls ( P < 0.002). Impaired mitochondrial electron transport was associated with high levels of superoxide production in the CalTG mice. Taken together, these data indicate that calcineurin signaling affects mitochondrial energetics and superoxide production. The excessive production of superoxide may contribute to the development of cardiac failure.
Domain-domain associations in cystic fibrosis transmembrane conductance regulator
Authors: Wang Wenlan , He Zhaoping , O'Shaughnessy Thomas J. , Rux John , Reenstra William W.
Published: May 2013

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione- S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic dig estion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.