bluuebunny/crossref_metadata_2025 huggingface dataset:
EGF receptor downregulation depends on a trafficking motif in the distal tyrosine kinase domain
Authors: Jones Stacie M. , Foreman Susan K. , Shank Brian B. , Kurten Richard C.
Published: May 2013

On binding to its receptor, epidermal growth factor (EGF) initiates a cascade of events leading to cell proliferation or differentiation. In addition, the EGF receptor itself is downregulated to attenuate mitogenic signaling. Downregulation occurs through trafficking of receptors to lysosomes, culminating in proteolytic destruction of both the receptor and ligand; however, endocytic sorting mechanisms that underlie lysosomal targeting remain obscure. The goal of this study was to explore one aspect of the molecular basis for ligand-induced lysosomal targeting and degradation of EGF receptors. In this study, we identify a tyrosine-leucine motif (954YLVI) that is essential for transit of ligand-receptor complexes to lysosomes. When this motif is mutated, HEK 293 cells expressing the mutant receptors demonstrate impaired lysosomal targeting and downregulation compared with wild-type receptors.954YLVI is highly conserved among EGF receptors from various mammalian and invertebrate species and is critical for receptor downregulation. We propose that 954YLVI works in concert with at least two additional regions within the EGF receptor cytoplasmic domain that are essential for efficiently targeting ligand-receptor complexes to the lysosome.
Hypoxia reduces expression and function of system A amino acid transporters in cultured term human trophoblasts
Authors: Nelson D. M. , Smith S. D. , Furesz T. C. , Sadovsky Y. , Ganapathy V. , Parvin C. A. , Smith C. H.
Published: May 2013

We tested the hypothesis that hypoxia diminishes the expression and transport of neutral amino acids by system A in full-term human trophoblasts. Cytotrophoblasts from normal human placentas were cultured in standard conditions of 20% O2 or in 1% and 3% O2 for 24 h before assay. Neutral amino acid transport for systems A, ASC, and L was assayed at 24 and 72 h by the cluster-tray technique. Hypoxia during the initial 24 h of culture reduced system A transport by 82% in 1% O2 and by 37% in 3% O2 ( P < 0.01) compared with standard conditions. Hypoxia during the latter 24 h of the 72 h in culture reduced system A transport by 55% in 1% O2 and by 20% in 3% O2 ( P < 0.05) compared with standard conditions at 72 h. Hypoxia (1% O2) also reduced total amino acid transport by 40% in the more differentiated syncytiotrophoblasts present at 72 h. Northern analysis of trophoblasts in standard conditions showed that subtypes of human amino acid transporter A (hATA1 and hATA2) were each expressed in cytotrophoblasts and syncytiotrophoblasts. Hypoxia decreased expression of hATA1 and hATA2 in both trophoblast phenotypes. We conclude that hypoxia downregulates system A transporter expression and activity in cultured human trophoblasts.
cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo
Authors: Ameen Nadia A. , Marino Christopher , Salas Pedro J. I.
Published: May 2013

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is regulated by cAMP-dependent vesicle traffic and exocytosis to the apical membrane in some cell types, but this has not been demonstrated in the intestinal crypt. The distribution of CFTR, lactase (control), and fluid secretion were determined in rat jejunum after cAMP activation in the presence of nocodazole and primaquine to disrupt vesicle traffic. CFTR and lactase were localized by immunofluorescence, and surface proteins were detected by biotinylation of enterocytes. Immunoprecipitates from biotinylated and nonbiotinylated cells were analyzed by streptavidin detection and immunoblots. Immunolocalization confirmed a cAMP-dependent shift of CFTR but not lactase from a subapical compartment to the apical surface associated with fluid secretion that was reduced in the presence of primaquine and nocodazole. Analysis of immunoblots from immunoprecipitates after biotinylation revealed a 3.8 ± 1.7-fold ( P < 0.005) increase of surface-exposed CFTR after vasoactive intestinal peptide (VIP). These measurements provide independent corroboration supporting a role for vesicle traffic in regulating CFTR and cAMP-induced fluid transport in the intestine.
Functional comparison of renal Na-K-Cl cotransporters between distant species
Authors: Gagnon Édith , Forbush Biff , Caron Luc , Isenring Paul
Published: May 2013

In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm2). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm2 in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of86Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm2of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.
Modulation of smooth muscle phenotype in vitro by homologous cell substrate
Authors: Tao F. , Chaudry S. , Tolloczko B. , Martin J. G. , Kelly S. M.
Published: May 2013

We have developed a novel cell culture system that supports the shortening of smooth muscle cells. Primary rat airway smooth muscle cells were plated on an ethanol-fixed, confluent monolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells on HCS were spindle shaped with a well-defined long axis, whereas cells grown on glass were larger and irregularly shaped. Smooth muscle-specific α-actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting in greater changes in cell area or length on average, but the maximal capacity of shortening of individual cells was similar between the groups. Unlike cells on glass, cells on HCS shortened to methacholine. HCS was reversible and persisted over several passages. Agonists stimulated intracellular Ca2+ oscillations in cells on HCS, whereas they elicited biphasic peak and plateau transients in cells on glass. HCS modulates smooth muscle cell phenotype in vitro.
Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins
Authors: Yoo James , Nichols Anthony , Mammen Joshua , Calvo Isabel , Song Jaekyung C. , Worrell Roger T. , Matlin Karl , Matthews Jeffrey B.
Published: May 2013

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-α, -ϵ, and -δ ( 34 ), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC-δ inhibitor röttlerin, implicating a novel isozyme, likely PKC-ϵ. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC-ϵ-dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex.
S-nitrosothiols inhibit uterine smooth muscle cell proliferation independent of metabolism to NO and cGMP formation
Authors: Cornwell Trudy L. , Ceaser Erin K. , Li Jie , Marrs Kevin L. , Darley-Usmar Victor M. , Patel Rakesh P.
Published: May 2013

S-nitrosothiols (RSNOs) are important mediators of nitric oxide (NO) biology. The two mechanisms that appear to dominate in their biological effects are metabolism leading to the formation of NO and S-nitrosation of protein thiols. In this study we demonstrate that RSNOs inhibit uterine smooth muscle cell proliferation independent of NO. The antiproliferative effects of NO on vascular smooth muscle are well defined, with the classic NO-dependent production of cGMP being demonstrated as the active pathway. However, less is known on the role of NO in mediating uterine smooth muscle cell function, a process that is important during menstruation and pregnancy. The RSNOs S-nitrosoglutathione and S-nitroso- N-acetyl pencillamine inhibited growth factor-dependent proliferation of human and rat uterine smooth muscle cells (ELT-3). Interestingly, these cells reduced RSNOs to generate NO. However, use of NO donors and other activators of the cGMP pathway failed to inhibit proliferation. These findings demonstrate the tissue-specific nature of responses to NO and demonstrate the presence of a RSNO-dependent but NO-independent pathway of inhibiting DNA synthesis in uterine smooth muscle cells.
Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death
Authors: Knight Martin M. , Roberts Susan R. , Lee David A. , Bader Dan L.
Published: May 2013

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2+ transients and oscillations. Decreasing the power of the laser light decreased the percentage of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was ∼90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells.
Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis
Authors: Gittens Joanne E. I. , Mhawi Abdul Amir , Lidington Darcy , Ouellette Yves , Kidder Gerald M.
Published: May 2013

Ovarian granulosa cells are coupled via gap junctions containing connexin43 (Cx43 or α-1 connexin). In the absence of Cx43, granulosa cells stop growing in an early preantral stage. However, the fact that granulosa cells of mature follicles express multiple connexins complicated interpretation of this finding. The present experiments were designed to clarify the role of Cx43 vs. these other connexins in the earliest stages of folliculogenesis. Dye injection experiments revealed that granulosa cells from Cx43 knockout follicles are not coupled, and this was confirmed by ionic current injections. Furthermore, electron microscopy revealed that gap junctions are extremely rare in mutant granulosa cells. In contrast, mutant granulosa cells were able to form gap junctions with wild-type granulosa cells in a dye preloading assay. It was concluded that mutant granulosa cells contain a population of connexons, composed of an unidentified connexin, that do not normally contribute to gap junctions. Therefore, although Cx43 is not the only gap junction protein present in granulosa cells of early preantral follicles, it is the only one that makes a significant contribution to intercellular coupling.
TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells
Authors: Jung Silke , Strotmann Rainer , Schultz Günter , Plant Tim D.
Published: May 2013

To investigate the possible role of members of the mammalian transient receptor potential (TRP) channel family (TRPC1–7) in vasoconstrictor-induced Ca2+ entry in vascular smooth muscle cells, we studied [Arg8]-vasopressin (AVP)-activated channels in A7r5 aortic smooth muscle cells. AVP induced an increase in free cytosolic Ca2+ concentration ([Ca2+]i) consisting of Ca2+ release and Ca2+ influx. Whole cell recordings revealed the activation of a nonselective cation current with a doubly rectifying current-voltage relation strikingly similar to those described for some heterologously expressed TRPC isoforms. The current was also stimulated by direct activation of G proteins as well as by activation of the phospholipase Cγ-coupled platelet-derived growth factor receptor. Currents were not activated by store depletion or increased [Ca2+]i. Application of 1-oleoyl-2-acetyl- sn-glycerol stimulated the current independently of protein kinase C, a characteristic property of the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents in A7r5 cells were increased by flufenamate. Northern hybridization revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that TRPC6 is a molecular component of receptor-stimulated Ca2+-permeable cation channels in A7r5 smooth muscle cells.
Bradykinin B1 receptor blocks PDGF-induced mitogenesis by prolonging ERK activation and increasing p27Kip1
Authors: Dixon Bradley S. , Evanoff David , Fang Wei B. , Dennis Michael J.
Published: May 2013

The mechanism by which the bradykinin B1 receptor (B1R) inhibits platelet-derived growth factor (PDGF)-stimulated proliferation was investigated in cultured rat mesenteric arterial smooth muscle cells. The B1R agonist des-Arg9-bradykinin (DABK) was found to inhibit PDGF-mediated activation of the cyclin E-cyclin-dependent kinase 2 (Cdk2) complex and to prevent hyperphosphorylation of retinoblastoma protein. DABK did not inhibit upregulation of cyclin E expression but increased expression of the Cdk2 inhibitor p27Kip1 and the association of p27Kip1 with the cyclin E-Cdk2 complex. In addition, DABK inhibited the PDGF-stimulated expression of cyclin D that would otherwise siphon p27Kip1 away from inhibition of cyclin E-Cdk2. The signaling mechanism by which DABK regulated p27Kip1 was explored. DABK was found to stimulate the activity of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) and to prolong activation of MEK and ERK by PDGF. Inhibition of ERK activation with the MEK inhibitors PD-98059 and U-0126 as well as the Src family kinase inhibitor PP2 completely blocked the effect of DABK to increase p27Kip1 and partially reversed the DABK-mediated inhibition of PDGF-stimulated proliferation. These studies demonstrate that the B1R inhibits PDGF-stimulated mitogenesis in part by prolonged activation of ERK leading to increased expression of p27Kip1.
Coregulation of fast contractile protein transgene and glycolytic enzyme expression in mouse skeletal muscle
Authors: Hallauer Patricia L. , Hastings Kenneth E. M.
Published: May 2013

Little is known of the gene regulatory mechanisms that coordinate the contractile and metabolic specializations of skeletal muscle fibers. Here we report a novel connection between fast isoform contractile protein transgene and glycolytic enzyme expression. In quantitative histochemical studies of transgenic mouse muscle fibers, we found extensive coregulation of the glycolytic enzyme glycerol-3-phosphate dehydrogenase (GPDH) and transgene constructs based on the fast skeletal muscle troponin I (TnIfast) gene. In addition to a common IIB > IIX > IIA fiber type pattern, TnIfast transgenes and GPDH showed correlated fiber-to-fiber variation within each fast fiber type, concerted emergence of high-level expression during early postnatal muscle maturation, and parallel responses to muscle under- or overloading. Regulatory information for GPDH-coregulated expression is carried by the TnIfast first-intron enhancer (IRE). These results identify an unexpected contractile/metabolic gene regulatory link that is amenable to further molecular characterization. They also raise the possibility that the equal expression in all fast fiber types observed for the endogenous TnIfast gene may be driven by different metabolically coordinated mechanisms in glycolytic (IIB) vs. oxidative (IIA) fast fibers.
Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle
Authors: Grover Ashok K. , Samson Sue E. , Robinson Sarah , Kwan Chiu Yin
Published: May 2013

Peroxynitrite generated in arteries from superoxide and NO may damage Ca2+ pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca2+ into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca2+ uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca2+ pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca2+ uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca2+ pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.
Attenuation of cAMP accumulation in adult rat cardiac fibroblasts by IL-1β and NO: role of cGMP-stimulated PDE2
Authors: Gustafsson Åsa B. , Brunton Laurence L.
Published: May 2013

Treatment of cultured adult rat cardiac fibroblasts with interleukin-1β (IL-1β) induces the inducible nitric oxide synthase (iNOS) expression, increases nitric oxide (NO) and cGMP production, and attenuates cAMP accumulation in response to isoproterenol by ∼50%. Reduced cAMP accumulation is due to NO production: the effect is mimicked by NO donors and prevented by N G-monomethyl-l-arginine, an NOS inhibitor. Effects of NO are not restricted to the β-adrenergic response; the response to forskolin is similarly diminished. NO donors only slightly (12%) decrease forskolin-stimulated adenylyl cyclase (AC) activity in cardiac fibroblast plasma membranes, suggesting that the main effect of NO is not a direct one on AC. An inhibitor of soluble guanylyl cyclase inhibits the effects of IL-1β and NO donors; inhibition of cGMP-dependent protein kinase is without effect. 3-Isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor, and erythro-9-(2-hydroxy-3-nonyl)adenine, a specific inhibitor of the cGMP-stimulated PDE (PDE2), completely restore cAMP accumulation in sodium nitroprusside-treated fibroblasts and largely reverse the attenuated response in IL-1β-treated fibroblasts. Although NO reportedly acts by reducing AC activity in some cells, in cardiac fibroblasts NO production decreases cAMP accumulation largely by the cGMP-mediated activation of PDE2.
Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells
Authors: Sherwood Jennifer J. , Eddinger Thomas J.
Published: May 2013

In smooth muscle cells (SMCs) isolated from rabbit carotid, femoral, and saphenous arteries, relative myosin isoform mRNA levels were measured in RT-PCR to test for correlations between myosin isoform expression and unloaded shortening velocity. Unloaded shortening velocity and percent smooth muscle myosin heavy chain 2 (SM2) and myosin light chain 17b (MLC17b) mRNA levels were not significantly different in single SMCs isolated from the luminal and adluminal regions of the carotid media. Saphenous artery SMCs shortened significantly faster ( P < 0.05) than femoral SMCs and had more SM2 mRNA ( P < 0.05) than carotid SMCs and less MLC17b mRNA ( P < 0.001) and higher tissue levels of SMB mRNA ( P < 0.05) than carotid and femoral SMCs. No correlations were found between percent SM2 and percent MLC17b mRNA levels and unloaded shortening velocity in SMCs from these arteries. We have previously shown that myosin heavy chain (MHC) SM1/SM2 and SMA/SMB and MLC17a/MLC17bisoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression and unloaded shortening velocity do not vary with distance from the lumen of the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/or MLC17a/MLC17b isoform expression does not correlate with unloaded shortening velocity, and 3) intracellular expression of the MHC SM1/SM2 and MLC17a/MLC17b isoforms is not coregulated.
Zinc depletion reduced Egr-1 and HNF-3β expression and apolipoprotein A-I promoter activity in Hep G2 cells
Authors: Cui Libin , Schoene Norberta W. , Zhu Lei , Fanzo Jessica C. , Alshatwi Ali , Lei K. Y.
Published: May 2013

We examined the influence of zinc status on expression of certain transcription factors involved in regulation of apolipoprotein A-I (apoAI) expression in human hepatoblastoma Hep G2 cells. A low zinc basal medium (zinc deficient, ZD) consisting of DMEM and 10% Chelex100-treated fetal bovine serum was used to deplete cellular zinc over one passage. Cells were also cultured for one passage in medium supplemented with 0.4 (ZD0.4), 4.0 (zinc normal, ZN), 16.0 (zinc adequate, ZA), or 32.0 μM zinc (zinc supplemented, ZS). Compared with ZN cells, cellular zinc levels were 43 and 31% lower in ZD and ZD0.4 cells but 70 and 146% higher in ZA and ZS cells, respectively. Supplementation of 0.4 μM zinc significantly increased DNA contents per plate, from 65% in ZD cells to 83% in ZD0.4 cells compared with ZN cells. Addition of >4 μM zinc in medium did not further increase DNA contents. The proportion of cells in G1/S and S phase was about fourfold higher and threefold lower, respectively, in ZD cells compared with ZN and other groups. Nuclear Egr-1 protein was markedly decreased in ZD and ZD0.4 cells. Moreover, hepatocyte nuclear factor (HNF)-3β was severely degraded in ZD and ZD0.4 cells. In contrast, HNF-4α remained stable in all groups and was not significantly lower in ZD and ZD0.4 cells. Furthermore, downregulation of trans-acting factor Egr-1 and cleavage of HNF-3β were associated with reduction of apoAI promoter activity in zinc-deficient Hep G2 cells. Thus zinc is critical in transcriptional regulation of apoAI gene expression in hepatocytes.
Tumor necrosis factor regulates intestinal epithelial cell migration by receptor-dependent mechanisms
Authors: Corredor Julissa , Yan Fang , Shen Christopher C. , Tong Wei , John Sutha K. , Wilson Guinn , Whitehead Robert , Polk D. Brent
Published: May 2013

Altered mucosal integrity and increased cytokine production, including tumor necrosis factor (TNF), are the hallmarks of inflammatory bowel disease (IBD). In this study, we addressed the role of TNF receptors (TNFR) on intestinal epithelial cell migration in an in vitro wound closure model. With mouse TNFR1 or TNFR2 knockout intestinal epithelial cells, gene transfection, and pharmacological inhibitors, we show a concentration-dependent receptor-mediated regulation of intestinal cell migration by TNF. A physiological TNF level (1 ng/ml) enhances migration through TNFR2, whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1 cannot be explained by either increased proliferation or apoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude that TNFR2 activates a novel Src-regulated pathway involving FAK tyrosine phosphorylation that enhances migration of intestinal epithelial cells.
V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration
Authors: Granger D. , Marsolais M. , Burry J. , Laprade R.
Published: May 2013

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO[Formula: see text]-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpHi/d t) following an acid load. Oligomycin (25 μM), an inhibitor of F-type ATPases, decreased dpHi/d t by 88 ± 6%, suggesting a role for an ATP-dependent process involved in pHi recovery. Moreover, dpHi/d t was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 μM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpHi/d t by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.
Targeted expression of activated Q227L Gαs in vivo
Authors: Huang Xi-Ping , Song Xiaosong , Wang Hsien-Yu , Malbon Craig C.
Published: May 2013

We report the creation of transgenic mice with an inducible, tissue-targeted expression of a constitutively active mutant form (Q227L) of Gαs. Mice expressing activated Gαs in fat tissue, liver, and skeletal muscle displayed normal body mass and blunted glucose metabolism. cAMP accumulation in adipose tissue was increased in the basal state, but far less than would be expected. Marked adaptation to elevated cAMP levels occurred, leading to an increase in total cAMP-specific phosphodiesterase activity, a 50% decline in cAMP-dependent protein kinase (protein kinase A) activity, and an increased expression of Gαi2. The reduction in kinase activity coincided with >50% increase in the expression of RIα and RIIα regulatory subunits of protein kinase A, with no change in the amount of catalytic subunit. These data demonstrate the existence of adaptive responses of protein kinase A, phosphodiesterase, and Gαi2 in tissues expressing constitutively active Gαs that may act to rectify the impact of increased cAMP accumulation.
Estrogen attenuates postexercise HSP70 expression in skeletal muscle
Authors: Paroo Zain , Dipchand Elizabeth S. , Noble Earl G.
Published: May 2013

Exercise has been demonstrated as a physiological inducer of heat shock protein (HSP)70. Many of the proposed signals of this response exhibit sexual dimorphism. Thus the present objectives were to determine whether HSP70 induction after exercise exhibits gender specificity and to elucidate the mechanisms underlying such a phenomenon. Postexercise HSP70 induction in skeletal muscle was greater in male than female rats at the level of protein and mRNA ( P = 0.005). Moreover, placebo-treated ovariectomized animals demonstrated a greater HSP70 response to exercise than those treated with estrogen ( P = 0.015 and 0.019 for protein and mRNA, respectively). These findings indicate that the gender-specific HSP70 response to exercise is mediated by the female-specific hormone estrogen. Compounds structurally related to 17β-estradiol, the major endogenous estrogen, but which do not activate the estrogen receptor, also attenuated HSP70 induction with exercise ( P < 0.01), indicating a nongenomic hormonal mechanism. These findings highlight a specific example of the biological differences between males and females and reiterate the physiological effects of sex hormones extending beyond their roles in reproductive function.
VACM-1, a cul-5 gene, inhibits cellular growth by a mechanism that involves MAPK and p53 signaling pathways
Authors: Van Dort C. , Zhao P. , Parmelee K. , Capps B. , Poel A. , Listenberger L. , Kossoris J. , Wasilevich B. , Murrey D. , Clare P. , Burnatowska-Hledin M.
Published: May 2013

Vasopressin-activated Ca2+-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression.
Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II
Authors: Gillis Todd E. , Moyes Chris D. , Tibbits Glen F.
Published: May 2013

Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca2+ than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca2+ sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca2+ affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca2+ affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca2+ binding to site II (F27W). The removal of the COOH terminus (amino acids 90–161) from ScTnC and McTnC maintained the difference in Ca2+ affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln29 and Asp30 in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca2+ affinity to that of McNTnC. These results demonstrate that Gln29 and Asp30 in ScTnC are required for the high Ca2+ affinity of site II.
Characterization of a voltage-dependent Na+current in human esophageal smooth muscle
Authors: Deshpande Maneesh A. , Wang Jian , Preiksaitis Harold G. , Laurier Lisanne G. , Sims Stephen M.
Published: May 2013

Smooth muscle contraction is critical to peristalsis in the human esophagus, yet the nature of the channels mediating excitation remains to be elucidated. The objective of this study was to characterize the inward currents in human esophageal smooth muscle cells (HESMCs). Esophageal tissue was isolated from patients undergoing surgery for cancer and grown in primary culture, and currents were recorded using patch-clamp electrophysiology. Depolarization elicited inward current activating positive to −40 mV and peaking at 0 mV and consisting of transient and sustained components. The transient current was half activated at −16 mV and half inactivated at −67 mV. The transient current was abolished by removal of bath Na+ or application of TTX (IC50∼20 nM), whereas it persisted in the absence of bath Ca2+or the presence of Cd2+. These data provide evidence that cultured HESMCs express voltage-dependent Na+ channels. RT-PCR revealed mRNA transcripts for Nax, the “atypical” Na+ channel isoform, as well as Nav1.4. These studies provide the first evidence of Nav1.4 in smooth muscle and contribute to a model of excitation in HESMCs.
Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation
Authors: Trimarchi James R. , Liu Lin , Smith Peter J. S. , Keefe David L.
Published: May 2013

Cell shrinkage is an incipient hallmark of apoptosis and is accompanied by potassium release that decreases the concentration of intracellular potassium and regulates apoptotic progression. The plasma membrane K+channel recruited during apoptosis has not been characterized despite its importance as a potential therapeutic target. Here we provide evidence that two-pore domain K+ (K2P) channels underlie K+ efflux during apoptotic volume decreases (AVD) in mouse embryos. These K2P channels are inhibited by quinine but are not blocked by an array of pharmacological agents that antagonize other K+ channels. The K2P channels are uniquely suited to participate in the early phases of apoptosis because they are not modulated by common intracellular messengers such as calcium, ATP, and arachidonic acid, transmembrane voltage, or the cytoskeleton. A K+channel with similar biophysical properties coordinates regulatory volume decreases (RVD) triggered by changing osmotic conditions. We propose that K2P channels are the pathway by which K+ effluxes during AVD and RVD and that apoptosis co-opts mechanisms more routinely employed for homeostatic cell volume regulation.
Interleukin-2 receptor-γ-dependent endocytosis depends on biotin in Jurkat cells
Authors: Rodriguez-Melendez Rocio , Camporeale Gabriela , Griffin Jacob B. , Zempleni Janos
Published: May 2013

Biotin has been credited with having beneficial effects on immune function despite observations that biotin supplementation causes decreased secretion of interleukin-2. Here this paradox was addressed by determining whether receptor-dependent internalization of interleukin-2 by immune cells depends on biotin. Theoretically, this would be consistent with both decreased net secretion of interleukin-2 by biotin-supplemented cells (causing increased endocytosis) and beneficial effects of biotin on immune function (causing increased receptor signaling). Jurkat cells were cultured in biotin-defined media (25, 250, or 10,000 pM). Secretion of interleukin-2 correlated negatively with biotin supply, but transcriptional activity of the interleukin-2 gene correlated positively with biotin supply, suggesting that decreased secretion of interleukin-2 by biotin-supplemented cells was not caused by decreased gene expression. Expression of the interleukin-2 receptor-γ gene was greater at 10,000 pM than 25 pM biotin, mediating increased endocytosis of interleukin-2 in biotin-supplemented medium. Inhibition of endocytosis by genistein and overexpression of interleukin-2 receptor-γ abolished the effect of biotin. These findings suggest that endocytosis of interleukin-2 depends on biotin.
Cholangiocytes exhibit dynamic, actin-dependent apical membrane turnover
Authors: Doctor R. Brian , Dahl Rolf , Fouassier Laura , Kilic Gordan , Fitz J. Gregory
Published: May 2013

The present studies of cholangiocytes used complementary histological, biochemical, and electrophysiological methods to identify a dense population of subapical vesicles, quantify the rates of vesicular trafficking, and assess the contribution of the actin cytoskeleton to membrane trafficking. FM 1–43 fluorescence measured significant basal rates of total exocytosis (1.33 ± 0.16% plasma membrane/min) in isolated cholangiocytes and apical exocytosis in cholangiocyte monolayers. Cell surface area remained unchanged, indicating that there was a concurrent, equivalent rate of endocytosis. FM 1–43 washout studies showed that 36% of the endocytosed membrane was recycled to the plasma membrane. 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP; cAMP analog) increased exocytosis by 71 ± 31%, whereas the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS; protein kinase A inhibitor) diminished basal exocytosis by 53 ± 11%. A dense population of 140-nm subapical vesicles arose, in part, from apical membrane endocytosis. Phalloidin staining showed that a subpopulation of the endocytosed vesicles was encapsulated by F-actin. Furthermore, membrane trafficking was inhibited by disrupting the actin cytoskeleton with cytochalasin D (51 ± 13% of control) or jasplakinolide (58 ± 9% of control). These studies indicate that there is a high rate of vesicular trafficking at the apical membrane of cholangiocytes and suggest that both cAMP and the actin cytoskeleton contribute importantly to these events.
Intracellular Cs+ activates the PKA pathway, revealing a fast, reversible, Ca2+-dependent inactivation of L-type Ca2+ current
Authors: Brette Fabien , Lacampagne Alain , Sallé Laurent , Findlay Ian , Le Guennec Jean-Yves
Published: May 2013

Inactivation of the L-type Ca2+ current ( ICaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, ICaL of 82% of cells infused with Cs+-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of ICaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased ICaL by only ∼60% in these cells. Cells infused with either N-methyl-d-glucamine or K+-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased ICaL by ∼200% in these cells. In conclusion, we show that multiphasic inactivation of ICaL is due to Ca2+-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs+ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K+ in the pipette solution.
Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice
Authors: Plant David R. , Lynch Gordon S.
Published: May 2013

Dystrophin is absent in muscle fibers of patients with Duchenne muscular dystrophy (DMD) and in muscle fibers from the mdx mouse, an animal model of DMD. Disrupted excitation-contraction (E-C) coupling has been postulated to be a functional consequence of the lack of dystrophin, although the evidence for this is not entirely clear. We used mechanically skinned fibers (with a sealed transverse tubular system) prepared from fast extensor digitorum longus muscles of wild-type control and dystrophic mdx mice to test the hypothesis that dystrophin deficiency would affect the depolarization-induced contractile response (DICR) and sarcoplasmic reticulum (SR) function. DICR was similar in muscle fibers from mdx and control mice, indicating normal voltage regulation of Ca2+ release. Nevertheless, rundown of DICR (<50% of initial) was reached more rapidly in fibers from mdx than control mice [control: 32 ± 5 depolarizations ( n = 14 fibers) vs. mdx: 18 ± 1 depolarizations ( n = 7) before rundown, P < 0.05]. The repriming rate for DICRs was decreased in fibers from mdx mice, with lower submaximal DICR observed after 5, 10, and 20 s of repriming compared with fibers from control mice ( P < 0.05). SR Ca2+ reloading was not different in fibers from control and mdx mice, and no difference was observed in SR Ca2+ leak. Caffeine (2–7 mM)-induced contraction was diminished in fibers from mdx mice compared with control ( P < 0.05), indicating depressed SR Ca2+ release channel activity. Our findings indicate that fast fibers from mdx mice exhibit some impairment in the events mediating E-C coupling and SR Ca2+ release channel activity.
Urate/α-ketoglutarate exchange in avian basolateral membrane vesicles
Authors: Grassl Steven M.
Published: May 2013

Membrane transport pathways for transcellular secretion of urate across the proximal tubule were investigated in avian kidney. The presence of coupled urate/α-ketoglutarate exchange was investigated in basolateral membrane vesicles (BLMV) by [14C]urate and [α-3H]ketoglutarate flux measurements. An inward Na gradient induced accumulation of α-ketoglutarate of sufficient magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na gradient also induced concentrative accumulation of urate in the presence of α-ketoglutarate but not in its absence, suggesting urate/α-ketoglutarate exchange. α-Ketoglutarate-dependent stimulation of urate uptake was not observed in brush-border membrane vesicles. An outward urate gradient induced concentrative accumulation of α-ketoglutarate. α-Ketoglutarate-coupled urate uptake was specific for α-ketoglutarate, Cl dependent, and insensitive to membrane potential. α-Ketoglutarate-coupled urate uptake was inhibited by increasing p-aminohippurate (PAH) concentrations, and α-ketoglutarate-coupled PAH uptake was observed. α-Ketoglutarate-coupled PAH uptake was inhibited by increasing urate concentrations, and an outward urate gradient induced concentrative accumulation of PAH. These results suggest a Cl-dependent, α-ketoglutarate-coupled anion exchange mechanism as a pathway for active urate uptake across the basolateral membrane of urate-secreting proximal tubule cells.
Role of nitric oxide in the regulation of HIF-1α expression during hypoxia
Authors: Agani Faton H. , Puchowicz Michelle , Chavez Juan Carlos , Pichiule Paola , LaManna Joseph
Published: May 2013

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1α and HIF-1β subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1α, is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1α hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1α accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1α hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1α accumulation during hypoxia. Regulation of HIF-1α by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.
Na+ pump α2-subunit expression modulates Ca2+ signaling
Authors: Golovina Vera A. , Song Hong , James Paul F. , Lingrel Jerry B. , Blaustein Mordecai P.
Published: May 2013

The role of the Na+ pump α2-subunit in Ca2+ signaling was examined in primary cultured astrocytes from wild-type (α2 +/+ = WT) mouse fetuses and those with a null mutation in one [α2 +/− = heterozygote (Het)] or both [α2 −/− = knockout (KO)] α2 genes. Na+ pump catalytic (α) subunit expression was measured by immunoblot; cytosol [Na+] ([Na+]cyt) and [Ca2+] ([Ca2+]cyt) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na+ pumps with both α1- (≈80% of total α) and α2- (≈20% of total α) subunits. Het astrocytes express ≈50% of normal α2; those from KO express none. Expression of α1 is normal in both Het and KO cells. Resting [Na+]cyt = 6.5 mM in WT, 6.8 mM in Het ( P > 0.05 vs. WT), and 8.0 mM in KO cells ( P < 0.001); 500 nM ouabain (inhibits only α2) equalized [Na+]cyt at 8 mM in all three cell types. Resting [Ca2+]cyt = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca2+ pumps and unloads the ER, induces transient (in Ca2+-free media) or sustained (in Ca2+-replete media) elevation of [Ca2+]cyt. These Ca2+ responses to 10 μM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca2+-free media, the reintroduction of Ca2+ induced significantly larger transient rises in [Ca2+]cyt (due to Ca2+ entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that α2 Na+ pumps and Na+/Ca2+ exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of α2 Na+ pump activity can elevate local [Na+] and, via Na+/Ca2+ exchange, [Ca2+] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca2+ stores and thereby amplifies Ca2+ signaling without elevating bulk [Na+]cyt.