bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells
Authors: Nakamura Ei'Ichiro , Uezono Yasuhito , Narusawa Ken'Ichiro , Shibuya Izumi , Oishi Yosuke , Tanaka Masahiro , Yanagihara Nobuyuki , Nakamura Toshitaka , Izumi Futoshi
Published: December 2017

In human osteoblast-like MG-63 cells, extracellular ATP increased [3H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [3H]thymidine incorporation. ATP-induced [3H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5′- O-(3-thiotriphosphate) and adenosine 5′-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [3H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid inhibited ATP-induced [3H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X4, P2X5, P2X6, and P2X7, but not P2X1, P2X2, and P2X3, receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca2+ concentration, and inhibitors for several Ca2+-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca2+ concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca2+-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.
Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells
Authors: Koh Timothy J. , Tidball James G.
Published: December 2017

We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C2C12 muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.
ATP and β-adrenergic stimulation enhance voltage-gated K current inactivation in brown adipocytes
Authors: Wilson Sean M. , Lee Sherwin C. , Shook Sheryl , Pappone Pamela A.
Published: December 2017

Sympathetic activation of brown fat thermogenesis stimulates adrenergic and purinergic receptors. We examined the effects of extracellular ATP and β-adrenergic agonists on voltage-activated K currents (IKv) in voltage-clamped rat brown adipocytes. ATP or the β-adrenergic agonist isoproterenol increased the development of IKv inactivation during depolarizing voltage steps in perforated patch-clamped cells. The effects on inactivation developed slowly in the presence of agonist and continued to increase for long times following agonist washout. 8-bromo-cAMP or forskolin had similar effects on IKv inactivation. Development of IKv inactivation during depolarizations was consistently enhanced by ATP or β-adrenergic stimulation in perforated-patch voltage-clamped cells but was not altered by these agents in whole cell recordings, suggesting that cytosolic factors are necessary for inactivation modulation. In either recording configuration, ATP or isoproterenol shifted the activation voltage dependence of IKv to more negative potentials, indicating the activation effect is mediated by a different pathway. Since both P2 purinergic and β-adrenergic signaling pathways generate fatty acids, we tested whether fatty acids could reproduce these modulations of IKv. Linoleic or arachidonic acid applied in whole cell recordings had effects similar to those of ATP or isoproterenol in perforated-patch experiments. These results are consistent with the possibility that β-adrenergic and P2 receptor stimulation modulate IKv through generation of fatty acids.
Sodium gradient-dependent transport of magnesium in rat ventricular myocytes
Authors: Tashiro Michiko , Konishi Masato
Published: December 2017

Cytoplasmic concentration of Mg2+([Mg2+]i) was measured with a fluorescent indicator furaptra in ventricular myocytes enzymatically dissociated from rat hearts (25°C). To study Mg2+ transport across the cell membrane, cells were treated with ionomycin in Ca2+-free (0.1 mM EGTA) and high-Mg2+ (10 mM) conditions to facilitate passive Mg2+ influx. Rate of rise of [Mg2+]i due to the net Mg2+influx was significantly smaller in the presence of 130 mM extracellular Na+ than in its absence. We also tested the extracellular Na+ dependence of the net Mg2+efflux from cells loaded with Mg2+. After [Mg2+]i was raised by ionomycin and high Mg2+ to the level 0.5–0.6 mM above the basal value (∼0.7 mM), washout of ionomycin and lowering extracellular [Mg2+] to 1.2 mM caused rapid decline of [Mg2+]i in the presence of 140 mM Na+. This net efflux of Mg2+ was completely inhibited by withdrawal of extracellular Na+ and was largely attenuated by imipramine, a known inhibitor of Na+/Mg2+ exchange, with 50% inhibition at 79 μM. The relation between the rate of net Mg2+ efflux and extracellular Na+ concentration ([Na+]o) had a Hill coefficient of 2 and [Na+]o at half-maximal rate of 82 mM. These results demonstrate the presence of Na+ gradient-dependent Mg2+ transport, which is consistent with Na+/Mg2+ exchange, in cardiac myocytes.
Species-specific difference in distribution of voltage-gated L-type Ca2+ channels of cardiac myocytes
Authors: Takagishi Yoshiko , Yasui Kenji , Severs Nicholas J. , Murata Yoshiharu
Published: December 2017

Ca2+influx via sarcolemmal voltage-dependent Ca2+ channels (L-type Ca2+ channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca2+ influx and intracellular Ca2+ release to activation of contraction. We investigated the distribution of L-type Ca2+ channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca2+ via surface sarcolemmal Ca2+ channels contributes to intracellular Ca2+ to a greater degree in rabbit than in rat myocytes.
Role of calcium and calmodulin in ciliary stimulation induced by acetylcholine
Authors: Zagoory Orna , Braiman Alex , Gheber Larisa , Priel Zvi
Published: December 2017

The goal of this work was to elucidate the molecular events underlying stimulation of ciliary beat frequency (CBF) induced by acetylcholine (ACh) in frog esophagus epithelium. ACh induces a profound increase in CBF and in intracellular Ca2+ concentration ([Ca2+]i) through M1 and M3 muscarinic receptors. The [Ca2+]i slowly decays to the basal level, while CBF stabilizes at an elevated level. These results suggest that ACh triggers Ca2+-correlated and -uncorrelated modes of ciliary stimulation. ACh response is abolished by the phospholipase C (PLC) inhibitor U-73122 and by depletion of intracellular Ca2+ stores but is unaffected by reduction of extracellular Ca2+ concentration and by blockers of Ca2+influx. Therefore, ACh activates PLC and mobilizes Ca2+solely from intracellular stores. The calmodulin inhibitors W-7 and calmidazolium attenuate the ACh-induced increase in [Ca2+]i but completely abolish the elevation in CBF. Therefore, elevation of [Ca2+]i is necessary for CBF enhancement but does not lead directly to it. The combined effect of Ca2+ elevation and of additional factors, presumably mobilized by Ca2+-calmodulin, results in a robust CBF enhancement.
Static stretch increases c-Jun NH2-terminal kinase activity and p38 phosphorylation in rat skeletal muscle
Authors: Boppart Marni D. , Hirshman Michael F. , Sakamoto Kei , Fielding Roger A. , Goodyear Laurie J.
Published: December 2017

Physical exercise and contraction increase c-Jun NH2-terminal kinase (JNK) activity in rat and human skeletal muscle, and eccentric contractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects of isometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus (EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats were subjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20–25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretch had a much more profound effect on the stress-activated protein kinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation of JNK occurred within the muscle fibers. These studies suggest that the stretch component of a muscle contraction may be a major contributor to the increases in JNK activity and p38 phosphorylation observed after exercise in vivo.
Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle
Authors: Sánchez-Ortiz Ricardo F. , Wang Ze , Menon Chandrakala , DiSanto Michael E. , Wein Alan J. , Chacko Samuel
Published: December 2017

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH2-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3′- or 5′-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls ( P < 0.04), which was reversed by estrogen replacement ( P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 × 106 copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively ( P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits ( P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits ( P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3′-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.
Muscarinic stimulation increases basal Ca2+ and inhibits spontaneous Ca2+ transients in murine colonic myocytes
Authors: Bayguinov Orline , Hagen Brian , Sanders Kenton M.
Published: December 2017

Localized Ca2+ transients in isolated murine colonic myocytes depend on Ca2+ release from inositol 1,4,5-trisphosphate (IP3) receptors. Localized Ca2+ transients couple to spontaneous transient outward currents (STOCs) and mediate hyperpolarization responses in these cells. We used confocal microscopy and whole cell patch-clamp recording to investigate how muscarinic stimulation, which causes formation of IP3, can suppress Ca2+ transients and STOCs that might override the excitatory nature of cholinergic responses. ACh (10 μM) reduced localized Ca2+ transients and STOCs, and these effects were associated with a rise in basal cytosolic Ca2+. These effects of ACh were mimicked by generalized rises in basal Ca2+ caused by ionomycin (250–500 nM) or elevated external Ca2+ (6 mM). Atropine (10 μM) abolished the effects of ACh. Pretreatment of cells with nicardipine (1 μM), or Cd2+ (200 μM) had no effect on responses to ACh. An inhibitor of phospholipase C, U-73122, blocked Ca2+ transients and STOCs but did not affect the increase in basal Ca2+ after ACh stimulation. Xestospongin C (Xe-C; 5 μM), a membrane-permeable antagonist of IP3 receptors, blocked spontaneous Ca2+ transients but did not prevent the increase of basal Ca2+ in response to ACh. Gd3+(10 μM), a nonselective cation channel inhibitor, prevented the increase in basal Ca2+ after ACh and increased the frequency and amplitude of Ca2+ transients and waves. Another inhibitor of receptor-mediated Ca2+ influx channels, SKF-96365, also prevented the rise in basal Ca2+after ACh and increased Ca2+ transients and development of Ca2+ waves. FK-506, an inhibitor of FKBP12/IP3 receptor interactions, had no effect on the rise in basal Ca2+ but blocked the inhibitory effects of increased basal Ca2+ and ACh on Ca2+transients. These results suggest that the rise in basal Ca2+ that accompanies muscarinic stimulation of colonic muscles inhibits localized Ca2+ transients that could couple to activation of Ca2+-activated K+channels and reduce the excitatory effects of ACh.
Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells
Authors: Lee Tzong-Shyuan , Chau Lee-Young
Published: December 2017

Oxidized low-density lipoprotein (oxLDL) is a potent inducer of apoptosis for vascular cells. In the present study, we demonstrate that the expression of death mediators, including p53, Fas, and Fas ligand (FasL) was substantially upregulated by oxLDL in cultured vascular smooth muscle cells (SMCs). The induction of these death mediators was time dependent and was accompanied by an increase in apoptotic death of SMCs following oxLDL treatment. Two oxysterols, 7β-hydroxycholesterol and 25-hydroxycholesterol, were also effective to induce the expression of death mediators and apoptosis. α-Tocopherol and deferoxamine significantly attenuated the induction of death mediators and cell death induced by oxLDL and oxysterols, suggesting that reactive oxygen species are involved in triggering the apoptotic event. Incubation of cells with FasL-neutralizing antibody inhibited the oxLDL-induced cell death up to 50%. Furthermore, caspase 8 and caspase 3 activities were induced time dependently in SMCs following oxLDL treatment. Collectively, these data suggest that the Fas/FasL death pathway is activated and responsible for, at least in part, the apoptotic death in vascular SMCs upon exposure to oxLDL.
Caloxin: a novel plasma membrane Ca2+ pump inhibitor
Authors: Chaudhary Jyoti , Walia Mandeep , Matharu Jaswinder , Escher Emanuel , Grover Ashok K.
Published: December 2017

Plasma membrane (PM) Ca2+ pump is a Ca2+-Mg2+-ATPase that expels Ca2+from cells to help them maintain low concentrations of cytosolic Ca2+. There are no known extracellularly acting PM Ca2+ pump inhibitors, as digoxin and ouabain are for Na+ pump. In analogy with digoxin, we define caloxins as extracellular PM Ca2+ pump inhibitors and describe caloxin 2A1. Caloxin 2A1 is a peptide obtained by screening a random peptide phage display library for binding to the second extracellular domain (residues 401–413) sequence of PM Ca2+ pump isoform 1b. Caloxin 2A1 inhibits Ca2+-Mg2+-ATPase in human erythrocyte leaky ghosts, but it does not affect basal Mg2+-ATPase or Na+-K+-ATPase in the ghosts or Ca2+-Mg2+-ATPase in the skeletal muscle sarcoplasmic reticulum. Caloxin 2A1 also inhibits Ca2+-dependent formation of the 140-kDa acid-stable acylphosphate, which is a partial reaction of this enzyme. Consistent with inhibition of the PM Ca2+ pump in vascular endothelium, caloxin 2A1 produces an endothelium-dependent relaxation that is reversed by N G-nitro-l-arginine methyl ester. Thus caloxin 2A1 is a novel PM Ca2+ pump inhibitor selected for binding to an extracellular domain.
cAMP-sensitive endocytic trafficking in A6 epithelia
Authors: Butterworth Michael B. , Helman Sandy I. , Els Willem J.
Published: December 2017

Blocker-induced noise analysis and laser scanning confocal microscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na+ channels (ENaCs) at apical membranes of A6 epithelia. After forskolin stimulation of Na+ transport and labeling apical membranes with the fluorescent dye N-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities ( N T) decreased exponentially (time constant ∼20 min) from mean values of 320 to 98 channels/cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled vesicles appeared in the cytosol within 55 min, reaching mean values near 18 vesicles/cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained within a few micrometers of the apical membranes for the duration of the experiments. A minority of vesicles migrated to >5 μm below the apical membrane. Because steady states require identical rates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at the apical membranes, but also principally to increase exocytic rates. These observations are consistent with and support, but do not prove, that vesicle trafficking is a mechanism for cAMP-mediated regulation of apical membrane channel densities in A6 epithelia.
Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells
Authors: Banerjee Abhijit , Li Guangmu , Alexander Edward A. , Schwartz John H.
Published: December 2017

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25–50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and35S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 ± 2% of control and reduces the rate of H+ secretion by 77 ± 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.
Induction of TNF-α and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4
Authors: Tsan Min-Fu , Clark Robert N. , Goyert Sanna M. , White Julie E.
Published: December 2017

Endotoxin (LPS) is a potent inducer of tumor necrosis factor-α (TNF-α) and manganese superoxide dismutase (MnSOD). Recent evidence suggests that LPS induction of TNF-α and MnSOD mRNAs is mediated through distinct intracellular signal transduction pathways. Membrane CD14 (mCD14) and Toll-like receptor-4 (TLR4) mediate LPS induction of TNF-α in macrophages. In the current study, we evaluated the role of mCD14 and TLR4 in LPS induction of MnSOD using peritoneal macrophages from CD14 knockout (CD14-KO) mice and mice with the Tlr4 gene point mutation (C3H/HeJ) or deletion (C57BL/10ScCr). We studied mCD14-dependent (1 and 10 ng/ml) and mCD14-independent (1,000 ng/ml) concentrations of LPS. Compared with control (BALB/c) macrophages, LPS at 1 and 10 ng/ml failed to induce TNF-α or MnSOD mRNA in CD14-KO macrophages. However, LPS at 1,000 ng/ml induced TNF-α and MnSOD mRNAs equally in macrophages from CD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induce TNF-α or MnSOD mRNA and failed to activate nuclear factor-κB in C3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF-α and MnSOD enzyme activity paralleled TNF-α and MnSOD mRNA levels. These data demonstrate that, like TNF-α, induction of MnSOD by LPS is mediated by mCD14 and TLR4 in murine macrophages.
Modulation of PKCδ tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases
Authors: Benes Cyril , Soltoff Stephen P.
Published: December 2017

Protein kinase C (PKC) δ becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCδ tyrosine phosphorylation and effects of phosphorylation on PKCδ activity. Carbachol- and substance P-promoted increases in PKCδ tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than d- myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCδ activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCδ activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCδ activation. Two PKCδ regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCδ tyrosine phosphorylation. These results demonstrate that PKCδ activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.
Contrasting effects of cPLA2 on epithelial Na+ transport
Authors: Worrell Roger T. , Bao Hui-Fang , Denson Don D. , Eaton Douglas C.
Published: December 2017

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+reabsorption in the cortical collecting duct. Xenopus laeviskidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2(PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.
Regions in the carboxy terminus of α-bENaC involved in gating and functional effects of actin
Authors: Copeland Susan J. , Berdiev Bakhrom K. , Ji Hong-Long , Lockhart Jason , Parker Suzanne , Fuller Catherine M. , Benos Dale J.
Published: December 2017

Gating differences occur between the α-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine α-ENaC (α-bENaC) at R567 converted the gating properties to that of rat α-ENaC (α-rENaC). The equivalent truncation in α-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either α-rENaC or α-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither α-rENaC (R613X) nor α-bENaC (R567X) was responsive to actin. Using a chimera consisting of α-rENaC1–615 and α-bENaC570–650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) α-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like α-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt α-bENaC. Key findings were confirmed using channels expressed in Xenopusoocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.
Localization of endogenous and recombinant Na+-driven anion exchanger protein NDAE1 fromDrosophila melanogaster
Authors: Sciortino Christopher M. , Shrode Lamara D. , Fletcher Bonnie R. , Harte Peter J. , Romero Michael F.
Published: December 2017

Na+-dependent Cl−/HCO[Formula: see text]exchange activity helps maintain intracellular pH (pHi) homeostasis in many invertebrate and vertebrate cell types. Our laboratory cloned and characterized a Na+-dependent Cl−/HCO[Formula: see text] exchanger (NDAE1) from Drosophila melanogaster (Romero MF, Henry D, Nelson S, Harte PJ, and Sciortino CM. J Biol Chem 275: 24552–24559, 2000). In the present study we used immunohistochemical and Western blot techniques to characterize the developmental expression, subcellular localization, and tissue distribution of NDAE1 protein in D. melanogaster. We have shown that a polyclonal antibody raised against the NH2terminus of NDAE1 (αCWR57) recognizes NDAE1 electrophysiologically characterized in Xenopus oocytes. Moreover, our results begin to delineate the NDAE1 topology, i.e., both the NH2and COOH termini are intracellular. NDAE1 is expressed throughout Drosophila development in the central and peripheral nervous systems, sensilla, and the alimentary tract (Malpighian tubules, gut, and salivary glands). Coimmunolabeling of larval tissues with NDAE1 antibody and a monoclonal antibody to the Na+-K+-ATPase α-subunit revealed that the majority of NDAE1 is located at the basolateral membranes of Malpighian tubule cells. These results suggest that NDAE1 may be a key pHi regulatory protein and may contribute to basolateral ion transport in epithelia and nervous system of Drosophila.
Factor Xa is a fibroblast mitogen via binding to effector-cell protease receptor-1 and autocrine release of PDGF
Authors: Blanc-Brude Olivier P. , Chambers Rachel C. , Leoni Patricia , Dik Willem A. , Laurent Geoffrey J.
Published: December 2017

The coagulation cascade protease thrombin is a fibroblast mitogen, but the proliferative potential of other coagulation proteases is not known. In this study we show that factor Xa stimulated human fetal lung fibroblast DNA synthesis in a concentration-dependent manner from 1 nM onward with a fourfold increase at 200 nM. The mitogenic effect of factor Xa was confirmed using a colorimetric proliferation assay and direct cell counting. Factor Xa and thrombin had equivalent potencies, and their stimulatory effects followed a similar time course. Comparable results were also obtained with primary human adult fibroblasts derived from lung, kidney, heart, skin, and liver. Factor VIIa also stimulated fibroblast proliferation, but only at concentrations >10 nM, whereas factor IXa had no effect. To begin to address the mechanism by which factor Xa is acting, we show that human fibroblasts express effector-cell protease receptor-1 and that blocking antibodies to this receptor and the catalytic site of factor Xa inhibited its mitogenic effect. Furthermore, factor Xa upregulated platelet-derived growth factor-A (PDGF-A) mRNA expression, whereas PDGF-B could not be detected, and a blocking antibody to PDGF inhibited the mitogenic effect of factor Xa. We conclude that factor Xa acts as a fibroblast mitogen via binding to effector-cell protease receptor-1 and the autocrine release of PDGF.
Enhanced mitochondrial testicular antioxidant capacity in Goto-Kakizaki diabetic rats: role of coenzyme Q
Authors: Palmeira Carlos M. , Santos Dario L. , Seiça Raquel , Moreno António J. , Santos Maria S.
Published: December 2017

Because diabetes mellitus is associated with impairment of testicular function, ultimately leading to reduced fertility, this study was conducted to evaluate the existence of a cause-effect relationship between increased oxidative stress in diabetes and reduced mitochondrial antioxidant capacity. The susceptibility to oxidative stress and antioxidant capacity (in terms of glutathione, coenzyme Q, and vitamin E content) of testis mitochondrial preparations isolated from Goto-Kakizaki (GK) non-insulin-dependent diabetic rats and from Wistar control rats, 1 yr of age, was evaluated. It was found that GK mitochondrial preparations showed a lower susceptibility to lipid peroxidation induced by ADP/Fe2+, as evaluated by oxygen consumption and reactive oxygen species generation. The decreased susceptibility to oxidative stress in diabetic rats was associated with an increase in mitochondrial glutathione and coenzyme Q9 contents, whereas vitamin E was not changed. These results demonstrate a higher antioxidant capacity in diabetic GK rats. We suggest this is an adaptive response of testis mitochondria to the increased oxidative damage in diabetes mellitus.
Role of phospholamban in the modulation of arterial Ca2+ sparks and Ca2+-activated K+ channels by cAMP
Authors: Wellman George C. , Santana L. F. , Bonev Adrian D. , Nelson Mark T.
Published: December 2017

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca2+-ATPase, and this inhibition is relieved by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. The role of PLB in regulating Ca2+ release through ryanodine-sensitive Ca2+ release channels, measured as Ca2+ sparks, was examined using smooth muscle cells of cerebral arteries from PLB-deficient (“knockout”) mice (PLB-KO). Ca2+ sparks were monitored optically using the fluorescent Ca2+ indicator fluo 3 or electrically by measuring transient large-conductance Ca2+-activated K+ (BK) channel currents activated by Ca2+sparks. Basal Ca2+ spark and transient BK current frequency were elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, an activator of adenylyl cyclase, increased the frequency of Ca2+ sparks and transient BK currents in cerebral arteries from control mice. However, forskolin had little effect on the frequency of Ca2+ sparks and transient BK currents from PLB-KO cerebral arteries. Forskolin or PLB-KO increased SR Ca2+ load, as measured by caffeine-induced Ca2+transients. This study provides the first evidence that PLB is critical for frequency modulation of Ca2+ sparks and associated BK currents by PKA in smooth muscle.
Ca2+ regulation of gap junctional coupling in lens epithelial cells
Authors: Churchill Grant C. , Lurtz Monica M. , Louis Charles F.
Published: December 2017

The quantitative effects of Ca2+signaling on gap junctional coupling in lens epithelial cells have been determined using either the spread of Mn2+ that is imaged by its ability to quench the fluorescence of fura 2 or the spread of the fluorescent dye Alexa Fluor 594. Gap junctional coupling was unaffected by a mechanically stimulated cell-to-cell Ca2+wave. Furthermore, when cytosolic Ca2+ concentration (Ca[Formula: see text]) increased after the addition of the agonist ATP, coupling was unaffected during the period that Ca[Formula: see text] was maximal. However, coupling decreased transiently ∼5–10 min after agonist addition when Ca[Formula: see text] returned to resting levels, indicating that this transient decrease in coupling was unlikely due to a direct action of Ca[Formula: see text] on gap junctions. An increase in Ca[Formula: see text] mediated by the ionophore ionomycin that was sustained for several minutes resulted in a more rapid and sustained decrease in coupling (IC50 ∼300 nM Ca2+, Hill coefficient of 4), indicating that an increase in Ca[Formula: see text]alone could regulate gap junctions. Thus Ca[Formula: see text]increases that occurred during agonist stimulation and cell-to-cell Ca2+ waves were too transient to mediate a sustained uncoupling of lens epithelial cells.
Linoleic acid both enhances activation and blocks Kv1.5 and Kv2.1 channels by two separate mechanisms
Authors: McKay M. Craig , Worley Jennings F.
Published: December 2017

Linoleic acid (LA) had two effects on human Kv1.5 and Kv2.1 channels expressed in Chinese hamster ovary cells: an increase in the speed of current activation process (EC50 = 2.4 and 2.7 μM for Kv1.5 and Kv2.1, respectively) and current inhibition (IC50 = 6.6 and 7.4 for Kv1.5 and Kv2.1, respectively). LA affected the activation kinetics via two processes: a leftward shift in the instantaneous activation curves and an increase in the rate of current rise. Current inhibition by LA was time dependent but voltage independent. Hill slopes for plots of current inhibition (3.5 and 3.9 for Kv1.5 and Kv2.1, respectively) vs. dose of LA suggested that cooperativity was involved in the mechanism of current inhibition. A similar analysis of the effects of LA on current activation did not reveal cooperative interactions. The effects of LA were mediated from the external side of the channels, since addition of 10 μM LA to the patch pipette solution was without effect. Additionally, the methyl ester of LA was effective at enhancing peak current and promoting channel activation for Kv1.5 and Kv2.1 without inducing significant current inhibition.
Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminal cytoplasmic domain
Authors: Stewart A. K. , Chernova M. N. , Kunes Y. Z. , Alper S. L.
Published: December 2017

The role of intracellular pH (pHi) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated 36Cl− efflux from Xenopus oocytes during imposed variation of extracellular pH (pHo) or variation of pHi at constant pHo. Wild-type AE2-mediated 36Cl−efflux displayed a steep pHo vs. activity curve, with pHo(50) = 6.91 ± 0.04. Sequential NH2-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pHo(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pHi at constant pHo and were shown to be neither substrates nor inhibitors of AE2-mediated Cl−transport. At constant pHo, AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH2-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.
Upregulation of α8β1-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-β1
Authors: Thibault Gaétan , Lacombe Marie-Josée , Schnapp Lynn M. , Lacasse Alexandre , Bouzeghrane Fatiha , Lapalme Geneviève
Published: December 2017

Using a novel pharmacological tool with125I-echistatin to detect integrins on the cell, we have observed that cardiac fibroblasts harbor five different RGD-binding integrins: α8β1, α3β1, α5β1, αvβ1, and αvβ3. Stimulation of cardiac fibroblasts by angiotensin II (ANG II) or transforming growth factor-β1 (TGF-β1) resulted in an increase of protein and heightening by 50% of the receptor density of α8β1-integrin. The effect of ANG II was blocked by an AT1, but not an AT2, receptor antagonist, or by an anti-TGF-β1 antibody. ANG II and TGF-β1 increased fibronectin secretion, smooth muscle α-actin synthesis, and formation of actin stress fibers and enhanced attachment of fibroblasts to a fibronectin matrix. The α8- and β1-subunits were colocalized by immunocytochemistry with vinculin or β3-integrin at focal adhesion sites. These results indicate that α8β1-integrin is an abundant integrin on rat cardiac fibroblasts. Its positive modulation by ANG II and TGF-β1 in a myofibroblast-like phenotype suggests the involvement of α8β1-integrin in extracellular matrix protein deposition and cardiac fibroblast adhesion.
Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea
Authors: Jayaraman Sujatha , Song Yuanlin , Verkman A. S.
Published: December 2017

Airway surface liquid (ASL) pH has been proposed to be important in the pathophysiology of cystic fibrosis, asthma, and cough. Ratio image analysis was used to measure pH in the ASL after staining with the fluorescent pH indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at an air-liquid interface was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of HCO[Formula: see text]/CO2. Steady-state ASL pH changed in parallel to changes in bath pH and was acidified by Na+ or Cl− replacement but was not affected by the inhibitors amiloride, glibenclamide, or 4,4′-dinitrostilbene-2,2′-disulfonic acid. In response to sudden acidification or alkalization of the ASL by ∼0.4 pH units by HCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pH units/min (−HCO[Formula: see text]) and 0.060 pH units/min (+HCO[Formula: see text]); the pH recovery rate was reduced by amiloride and H2DIDS. In anesthetized mice in which the trachea was surgically exposed for measurement of BCECF-dextran fluorescence through the translucent tracheal wall, ASL pH was 7.14 ± 0.01. ASL pH was sensitive to changes in blood pH created by metabolic (HCl or NaHCO3 infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, and H+/OH− transport between the ASL and basolateral fluid involves amiloride-sensitive Na+/H+ exchange and stilbene-sensitive Cl−/HCO[Formula: see text] exchange. The rapid response of ASL pH to changes in systemic acid-base status may contribute to airway hypersensitivity in asthma and other airway diseases.
Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose
Authors: Malhotra Ricky , Lin Zhiwu , Vincenz Claudius , Brosius Frank C.
Published: December 2017

Glucose uptake and metabolism inhibit hypoxia-induced apoptosis in a variety of cell types, but the underlying molecular mechanisms remain poorly understood. In the present study, we explore hypoxia-mediated cell death pathways in Jurkat cells in the presence and absence of extracellular glucose. In the absence of extracellular glucose, hypoxia caused cytochrome c release, caspase 3 and poly(ADP-ribose)polymerase cleavage, and DNA fragmentation; this apoptotic response was blocked by the caspase 9 inhibitor z-LEHD-FMK. The presence of extracellular glucose during hypoxia prevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In these conditions, overexpression of the caspase 8 inhibitor v-FLIP prevented hypoxia-mediated cell death. Thus hypoxia can stimulate two apoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucose uptake and metabolism, and the other independent of cytochrome c release and resulting from activation of the death receptor pathway, which is accelerated by glucose uptake and metabolism.
A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy
Authors: Mitchell Patrick O. , Pavlath Grace K.
Published: December 2017

Slow-twitch skeletal muscle atrophies greatly in response to unloading conditions. The cellular mechanisms that contribute to the restoration of muscle mass after atrophy are largely unknown. Here, we show that atrophy of the mouse soleus is associated with a 36% decrease in myonuclear number after 2 wk of hindlimb suspension. Myonuclear number is restored to control values during the 2-wk recovery period in which muscle mass returns to normal, suggesting that muscle precursor cells proliferate and fuse with myofibers. Inhibition of muscle precursor cell proliferation by local γ-irradiation of the hindlimb completely prevents this increase in myonuclear number. Muscle growth occurs normally during the first week in irradiated muscles, but growth during the second week is inhibited, leading to a 50% attenuation in the restoration of muscle mass. Thus early muscle growth occurs independently of an increase in myonuclear number, whereas later growth requires proliferating muscle precursor cells leading to myonuclear accretion. These results suggest that increasing the proliferative capacity of muscle precursor cells may enhance restoration of muscle mass after atrophy.
Regulation of cardiac and smooth muscle Ca2+channels (CaV1.2a,b) by protein kinases
Authors: Keef Kathleen D. , Hume Joseph R. , Zhong Juming
Published: December 2017

High voltage-activated Ca2+ channels of the CaV1.2 class (L-type) are crucial for excitation-contraction coupling in both cardiac and smooth muscle. These channels are regulated by a variety of second messenger pathways that ultimately serve to modulate the level of contractile force in the tissue. The specific focus of this review is on the most recent advances in our understanding of how cardiac CaV1.2a and smooth muscle CaV1.2b channels are regulated by different kinases, including cGMP-dependent protein kinase, cAMP-dependent protein kinase, and protein kinase C. This review also discusses recent evidence regarding the regulation of these channels by protein tyrosine kinase, calmodulin-dependent kinase, purified G protein subunits, and identification of possible amino acid residues of the channel responsible for kinase regulation.
Cloning and functional characterization of a new subtype of the amino acid transport system N
Authors: Nakanishi Takeo , Kekuda Ramesh , Fei You-Jun , Hatanaka Takahiro , Sugawara Mitsuru , Martindale Robert G. , Leibach Frederick H. , Prasad Puttur D. , Ganapathy Vadivel
Published: December 2017

We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50–56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na+-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li+tolerant, and pH sensitive. The transport mechanism involves the influx of Na+ and amino acids coupled to the efflux of H+, resulting in intracellular alkalization. Proline, α-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.
Regulation of K+ current in human airway epithelial cells by exogenous and autocrine adenosine
Authors: Szkotak Artur J. , Ng Amy M. L. , Sawicka Jolanta , Baldwin Stephen A. , Man S. F. Paul , Cass Carol E. , Young James D. , Duszyk Marek
Published: December 2017

The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K+ channels, most likely Ca2+-dependent intermediate-conductance K+ ( I K) channels. A1 but not A2 receptor antagonists blocked the effects of adenosine. RT-PCR studies showed that A549 cells expressed mRNA for I K-1 channels as well as A1, A2A, and A2B but not A3 receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of K+ channels in A549 cells and that hENTs play a crucial role in this process.