bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential distribution of ERα and ERβ mRNA in intrauterine tissues of the pregnant rhesus monkey
Authors: Wu Wen Xuan , Ma Xiao Hong , Smith Gordon C. S. , Nathanielsz Peter W.
Published: December 2017

Two estrogen receptor (ER) isoforms, ERα and ERβ, have been described. However, no information is available in any species regarding the comparison of ERα and ERβ levels in pregnant intrauterine tissues. We investigated 1) distribution of ERα and ERβ mRNA in myometrium, amnion, choriodecidua, and placenta; 2) their abundance in intrauterine tissues at term not in labor (NIL) and in spontaneous term labor (STL); and 3) immunolocalization of ERα and ERβ in pregnant rhesus monkey myometrium. Myometrium, amnion, choriodecidua, and placenta were obtained at cesarean section from monkeys in STL at 156–166 days gestational age (GA) ( n = 4) and from control monkeys NIL at 140–152 days GA ( n = 4). RT-PCR was conducted to determine ERα and ERβ and glyceraldehyde-3-phosphate dehydrogenase mRNA abundance in four intrauterine tissues of the pregnant rhesus monkey. The cloned ERβ PCR fragment was subjected to sequence analysis. ERα and ERβ were localized in the myometrium by immunohistochemistry. We demonstrated that 1) rhesus monkey ERβ shares >97% identity with human ERβ in the region sequenced; 2) both ERs were expressed in myometrium, amnion, and choriodecidua but not in placenta in the current study; 3) ERα and ERβ were differentially distributed in myometrium and amnion; 4) ERα and ERβ were immunolocalized in myometrial smooth cells and smooth muscle and endothelial cells of the myometrial blood vessels. The biological significance of these quantitative differences in ER subtypes merits further study.
RPTPμ and protein tyrosine phosphorylation regulate K+ channel mRNA expression in adult cardiac myocytes
Authors: Hershman Kenneth M. , Levitan Edwin S.
Published: December 2017

Previously, we reported that cell-cell contact regulates K+ channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase μ (RPTPμ) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPμ. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K+ channel genes. Furthermore, we conclude that RPTPμ specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.
Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing
Authors: Lee Hsinyu , Goetzl Edward J. , An Songzhu
Published: December 2017

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent lipid growth factors with similar abilities to stimulate cytoskeleton-based cellular functions. Their effects are mediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes ( edgs). We hypothesize that large quantities of LPA and S1P generated by activated platelets may influence endothelial cell functions. Using an in vitro wound healing assay, we observed that LPA and S1P stimulated closure of wounded monolayers of human umbilical vein endothelial cells and adult bovine aortic endothelial cells, which express LPA receptor Edg2, and S1P receptors Edg1 and Edg3. The two major components of wound healing, cell migration and proliferation, were stimulated individually by both lipids. LPA and S1P also stimulated intracellular Ca2+mobilization and mitogen-activated protein kinase (MAPK) phosphorylation. Pertussis toxin partially blocked the effects of both lipids on endothelial cell migration, MAPK phosphorylation, and Ca2+ mobilization, implicating Gi/o-coupled Edg receptor signaling in endothelial cells. LPA and S1P did not cross-desensitize each other in Ca2+ responses, suggesting involvement of distinct receptors. Thus LPA and S1P affect endothelial cell functions through signaling pathways activated by distinct GPCRs and may contribute to the healing of wounded vasculatures.
Eicosatetraynoic and eicosatriynoic acids, lipoxygenase inhibitors, block meiosis via antioxidant action
Authors: Takami M. , Preston S. L. , Behrman H. R.
Published: December 2017

We previously showed that nordihydroguaiaretic acid (NDGA) and other antioxidants inhibit the resumption of meiosis in oocyte-cumulus complexes (OCC) and denuded oocytes (DO). Because NDGA is well known to be an inhibitor of lipoxygenases (LOX), we assessed whether other LOX inhibitors influence spontaneous germinal vesicle breakdown (GVBD) in OCC and DO. Spontaneous GVBD in rat OCC obtained from preovulatory follicles was significantly and reversibly inhibited by the minimum effective doses of 80 and 100 μM 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,8,11-eicosatriynoic acid (ETI), respectively. In DO, GVBD was significantly inhibited by 100 μM ETYA or ETI. The minimum effective concentrations of ETYA and ETI for inhibition of GVBD in either OCC or DO are ∼30- to 50-fold higher than the concentrations necessary to inhibit LOX activity by 50% in intact cells. Because we previously showed that NDGA and other antioxidants inhibit the spontaneous resumption of meiosis, we assessed whether ETYA and ETI may act similarly as scavengers of reactive oxygen species (ROS). Luminol-amplified chemiluminescence showed that 50 μM of either ETYA or ETI markedly and significantly reduced ROS generated with 10 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH). Moreover, incubation of DO with 30 mM AAPH reversed the inhibition of GVBD produced by 100 μM ETYA or ETI. These findings support the conclusion that ETYA and ETI inhibit oocyte maturation by acting as antioxidants rather than by inhibiting LOX.
Mxi2, a splice variant of p38 stress-activated kinase, is a distal nephron protein regulated with kidney ischemia
Authors: Faccio Lucia , Chen Ang , Fusco Carlo , Martinotti Stefano , Bonventre Joseph V. , Zervos Antonis S.
Published: December 2017

Mxi2 is one of three known alternative spliced forms of the stress-activated mitogen-activated protein kinase p38 (CSBP). Mxi2 was originally identified as a Max-interacting protein and is the smallest member of the family of stress-activated kinases isolated to date. Mxi2 lacks most of the XI domain found in p38 and instead has a distinct COOH-terminal sequence of 17 amino acids. Here we present the genomic structure of the Mxi2/p38 locus on human chromosome 6q21.2/21.3 and establish the origin of the three spliced forms of p38. Using Mxi2-specific antibodies in mouse organs, we found the Mxi2 protein to be present exclusively in the kidney. Mxi2 is present predominantly in the distal tubule of the nephron and the level of the protein decreased during kidney ischemia-reperfusion. Stress signals or other known activators of the p38 pathway including MAP kinase-kinase 3 and MAP kinase-kinase 6 did not induce the kinase activity of Mxi2 using ATF-2 as a substrate. With the use of hybrid proteins encoding different portions of Mxi2 and p38 polypeptides, the different properties of Mxi2 can be assigned to its unique COOH terminus.
Cellular mechanisms involved in carotid body inhibition produced by atrial natriuretic peptide
Authors: He L. , Dinger B. , Fidone S.
Published: December 2017

Atrial natriuretic peptide (ANP) and its analog, atriopeptin III (APIII), inhibit carotid body chemoreceptor nerve activity evoked by hypoxia. In the present study, we have examined the hypothesis that the inhibitory effects of ANP and APIII are mediated by cyclic GMP and protein kinase G (PKG) via the phosphorylation and/or dephosphorylation of K+ and Ca2+ channel proteins that are involved in regulating the response of carotid body chemosensory type I cells to low-O2 stimuli. In freshly dissociated rabbit type I cells, we examined the effects of a PKG inhibitor, KT-5823, and an inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), on K+ and Ca2+ currents. We also investigated the effects of these specific inhibitors on intracellular Ca2+ concentration and carotid sinus nerve (CSN) activity under normoxic and hypoxic conditions. Voltage-dependent K+ currents were depressed by hypoxia, and this effect was significantly reduced by 100 nM APIII. The effect of APIII on this current was reversed in the presence of either 1 μM KT-5823 or 100 nM OA. Likewise, these drugs retarded the depression of voltage-gated Ca2+ currents induced by APIII. Furthermore, APIII depressed hypoxia-evoked elevations of intracellular Ca2+, an effect that was also reversed by OA and KT-5823. Finally, CSN activity evoked by hypoxia was decreased in the presence of 100 nM APIII, and was partially restored when APIII was presented along with 100 nM OA. These results suggest that ANP initiates a cascade of events involving PKG and PP2A, which culminates in the dephosphorylation of K+ and Ca2+ channel proteins in the chemosensory type I cells.
Normalization of hyperosmotic-induced inositol uptake by renal and endothelial cells is regulated by NF-κB
Authors: Yorek Mark A. , Dunlap Joyce A. , Liu Wenli , Lowe William L.
Published: December 2017

Hyperosmolarity is a stress factor that has been shown to cause an increase in the transcription of the Na+-dependent myo-inositol cotransporter (SMIT). However, regulation of the reversion of SMIT mRNA levels and transporter activity following removal of hyperosmotic stress is less understood. Previously we have shown that postinduction normalization of SMIT mRNA levels and myo-inositol accumulation following removal of hyperosmotic stress is inhibited by actinomycin D and cycloheximide, suggesting that normalization requires RNA transcription and protein synthesis. We now demonstrate that removal of hyperosmotic stress causes an activation of the transcription factor NF-κB in renal and endothelial cells. Inhibiting NF-κB activation with pyrrolidine dithiocarbamate (PD) blocks the normalization of SMIT mRNA levels and myo-inositol accumulation on removal of the cells from hyperosmotic medium. These studies demonstrate that the downregulation of the myo-inositol transporter following reversal of hyperosmotic induction is regulated via the activation of NF-κB.
Ca2+ and p38 MAP kinase regulate mAChR-mediated c-Fos expression in avian exocrine cells
Authors: Hildebrandt Jan-Peter , Prowald Alexandra
Published: December 2017

Muscarinic acetylcholine receptors (mAChRs) in exocrine tissue from the avian nasal salt gland are coupled to phospholipase C and generate inositol phosphate and Ca2+ signals upon activation. An early effect of receptor activation in the secretory cells is a transient accumulation of c-Fos protein. In cooperation with constitutively expressed Jun, Fos presumably serves as a transcription factor altering gene expression during cell growth and differentiation processes in the gland associated with adaptation to osmotic stress in animals. Nothing is known, however, about the mAChR-dependent signaling pathways leading to Fos expression in these cells. By incubation of isolated nasal gland tissue in short-term culture with activators or inhibitors of signaling pathways and quantitative Western blot analysis of Fos abundance, we have now identified the sustained elevation of the intracellular Ca2+ concentration and the activation of the p38 mitogen-activated protein (MAP) kinase as intermediate signaling elements for the regulation of c-Fos by muscarinic receptor activation. It is suggested that p38 MAP kinase, rather than exclusively mediating stress responses, is involved in the regulation of cellular growth and differentiation controlled by G protein-coupled receptors.
Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC
Authors: Bertog Marko , Smith David J. , Bielfeld-Ackermann Andreas , Bassett John , Ferguson David J. P. , Korbmacher Christoph , Harris Ann
Published: December 2017

To investigate the biology of the male genital duct epithelium, we have established cell cultures from the ovine vas deferens and epididymis epithelium. These cells develop tight junctions, high transepithelial electrical resistance, and a lumen-negative transepithelial potential difference as a sign of active transepithelial ion transport. In epididymis cultures the equivalent short-circuit current ( I sc) averaged 20.8 ± 0.7 μA/cm2 ( n = 150) and was partially inhibited by apical application of amiloride with an inhibitor concentration of 0.64 μM. In vas deferens cultures, I sc averaged 14.4 ± 1.1 μA/cm2 ( n = 18) and was also inhibited by apical application of amiloride with a half-maximal inhibitor concentration ( K i) of 0.68 μM. The remaining amiloride-insensitive I sc component in epididymis and vas deferens cells was partially inhibited by apical application of the Cl− channel blocker diphenylamine-2-carboxylic acid (1 mM). It was largely dependent on extracellular Cl− and, to a lesser extent, on extracellular[Formula: see text]. It was further stimulated by basolateral application of forskolin (10− 5 M), which increased I sc by 3.1 ± 0.3 μA/cm2 ( n=65) in epididymis and 0.9 ± 0.1 μA/cm2 ( n = 11) in vas deferens. These findings suggest that cultured ovine vas deferens and epididymis cells absorb Na+ via amiloride-sensitive epithelial Na+ channels (ENaC) and secrete Cl− and [Formula: see text]via apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. This interpretation is supported by RT-PCR data showing that vas deferens and epididymis cells express CFTR and ENaC mRNA.
Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency
Authors: Gow Andrew J. , Chen Qiping , Gole Madhura , Themistocleous Marios , Lee Virginia M.-Y. , Ischiropoulos Harry
Published: December 2017

To better understand the mechanism(s) underlying nitric oxide (⋅ NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing ⋅ NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25–750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for ⋅ NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after ⋅ NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not ⋅ NO-exposed cells. Cell injury from ⋅ NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-⋅ NO-producing cells to ⋅ NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.
Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole
Authors: Luo Jiexin , Zhu Tang , Evagelidis Alexandra , Pato Mary D. , Hanrahan John W.
Published: December 2017

Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [32P]PO4 release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)- p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate.
Osmotically relevant membrane signaling complex: association between HB-EGF, β1-integrin, and CD9 in mTAL
Authors: Sheikh-Hamad David , Youker Keith , Truong Luan D. , Nielsen Soren , Entman Mark L.
Published: December 2017

The integral membrane proteins cluster of differentiation-9 (CD9), β1-integrin, and heparin-binding epidermal growth factor-like (HB-EGF) exist in association in many cell lines and are linked to intracellular signaling mechanisms. Two of the proteins (CD9 and β1-integrin) are induced by hypertonicity, suggesting that their related signaling processes may be relevant to osmotic stress. The validity of this hypothesis rests upon coexpression and physical association between these molecules in nephron segments that are normally exposed to high and variable ambient osmolality. In this work, we show that CD9 and β1-integrin are induced in rat kidney medulla after dehydration. Immunohistochemistry and immunoprecipitation studies show that CD9, HB-EGF, and β1-integrin are coexpressed and physically associated in medullary thick ascending limbs (mTAL), nephron segments that are normally exposed to high and variable extracellular osmolality. Our findings are consistent with the existence of a cluster of integral membrane proteins in mTAL that may initiate or modulate osmotically relevant signaling pathways.
Mechanical strain-induced Ca2+waves are propagated via ATP release and purinergic receptor activation
Authors: Sauer H. , Hescheler J. , Wartenberg M.
Published: December 2017

Mechanical strain applied to prostate cancer cells induced an intracellular Ca2+ (Cai 2+) wave spreading with a velocity of 15 μm/s. Cai 2+ waves were not dependent on extracellular Ca2+ and membrane potential because propagation was unaffected in high-K+ and Ca2+-free solution. Waves did not depend on the cytoskeleton or gap junctions because cytochalasin B and nocodazole, which disrupt microfilaments and microtubules, respectively, and 1-heptanol, which uncouples gap junctions, were without effects. Fluorescence recovery after photobleaching experiments revealed an absence of gap junctional coupling. Cai 2+ waves were inhibited by the purinergic receptor antagonists basilen blue and suramin; by pretreatment with ATP, UTP, ADP, UDP, 2-methylthio-ATP, and benzoylbenzoyl-ATP; after depletion of ATP by 2-deoxyglucose; and after ATP scavenging by apyrase. Waves were abolished by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid, tamoxifen, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, niflumic acid, and gadolinium. ATP release following strain was significantly inhibited by anion channel blockers. Hence, ATP is secreted via mechanosensitive anion channels and activates purinergic receptors on the same cell or neighboring cells in an autocrine and paracrine manner, thus leading to Cai 2+ wave propagation.
Expression of GFRα-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB
Authors: Utsumi Hiroyuki , Chiba Hideki , Kamimura Yasuhiro , Osanai Makoto , Igarashi Yo , Tobioka Hirotoshi , Mori Michio , Sawada Norimasa
Published: December 2017

It is well known that the blood-brain barrier (BBB) matures at ∼2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRα-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRα-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c- ret, which is thought to mediate a signal evoked by binding of GDNF to GFRα-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRα-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.
Clathrin in mitotic spindles
Authors: Okamoto Curtis T. , McKinney Jeana , Jeng Young Y.
Published: December 2017

Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.
Similarity of A3-adenosine and swelling-activated Cl− channels in nonpigmented ciliary epithelial cells
Authors: Carré David A. , Mitchell Claire H. , Peterson-Yantorno Kim , Coca-Prados Miguel , Civan Mortimer M.
Published: December 2017

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl− transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A3-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)- N-methyl-β-d-ribofuranuronamide (IB-MECA) stimulated currents ( I IB-MECA) by ∼90% at +80 mV. Partial replacement of external Cl−with aspartate reduced outward currents and shifted the reversal potential ( V rev) from −23 ± 2 mV to −0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl− replacement with aspartate and NO3 −, and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents ( I Swell). Partial cyclamate substitution for external Cl− inhibited inward and outward currents of both I IB-MECA and I Swell. Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A3-subtype adenosine agonists and swelling activate a common population of Cl− channels.
Vibrio cholerae ACE stimulates Ca2+-dependent Cl−/HCO3 −secretion in T84 cells in vitro
Authors: Trucksis Michele , Conn Timothy L. , Wasserman Steven S. , Sears Cynthia L.
Published: December 2017

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current ( I sc) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I sc and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl− secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I sc and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl−and HCO3 −. ACE acted synergistically with the Ca2+-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca2+ but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca2+ as a second messenger and that this toxin stimulates a novel Ca2+-dependent synergy.
Acute glucose-induced downregulation of PKC-βII accelerates cultured VSMC proliferation
Authors: Yamamoto Mayumi , Acevedo-Duncan Mildred , Chalfant Charles E. , Patel Niketa A. , Watson James E. , Cooper Denise R.
Published: December 2017

Accelerated vascular smooth muscle cell (VSMC) proliferation contributes to the formation of atherosclerotic lesions. To investigate protein kinase C (PKC)-βII functions with regard to glucose-induced VSMC proliferation, human VSMC from aorta (AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cells overexpressing PKC-βI (βI-A10) and PKC-βII (βII-A10) were studied with the use of three techniques to evaluate glucose effects on aspects affecting proliferation. High glucose (25 mM) increased DNA synthesis and accelerated cell proliferation compared with normal glucose (5.5 mM) in AoSMC and A10 cells, but not in βI-A10 and βII-A10 cells. The PKC-βII specific inhibitor CGP-53353 inhibited glucose-induced cell proliferation and DNA synthesis in AoSMC and A10 cells. In flow cytometry analysis, high glucose increased the percentage of A10 cells at 12 h after cell cycle initiation but did not increase the percentage of βI-A10 or βII-A10 cells entering S phase. PKC-βII protein levels decreased before the peak of DNA synthesis, and high glucose further decreased PKC-βII mRNA and protein levels in AoSMC and A10 cells. These results suggest that high glucose downregulates endogenous PKC-βII, which then alters the normal inhibitory role of PKC-βII in cell cycle progression, resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.
Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca2+ channel currents
Authors: Mitarai Sayaka , Kaibara Muneshige , Yano Katsusuke , Taniyama Kohtaro
Published: December 2017

We investigated the inactivation process of macroscopic cardiac L-type Ca2+ channel currents using the whole cell patch-clamp technique with Na+ as the current carrier. The inactivation process of the inward currents carried by Na+ through the channel consisted of two components >0 mV. The time constant of the faster inactivating component (30.6 ± 2.2 ms at 0 mV) decreased with depolarization, but the time constant of the slower inactivating component (489 ± 21 ms at 0 mV) was not significantly influenced by the membrane potential. The inactivation process in the presence of isoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currents and attenuated the effects of isoproterenol. In the presence of cAMP (500 μM), the inactivation process consisted of a single slow component. We propose that the faster inactivating component represents a kinetic of the dephosphorylated or partially phosphorylated channel, and phosphorylation converts the kinetics into one with a different voltage dependency.
Calpain activity in fast, slow, transforming, and regenerating skeletal muscles of rat
Authors: Sultan Karim R. , Dittrich Bernd T. , Pette Dirk
Published: December 2017

Fiber-type transitions in adult skeletal muscle induced by chronic low-frequency stimulation (CLFS) encompass coordinated exchanges of myofibrillar protein isoforms. CLFS-induced elevations in cytosolic Ca2+ could activate proteases, especially calpains, the major Ca2+-regulated cytosolic proteases. Calpain activity determined by a fluorogenic substrate in the presence of unaltered endogenous calpastatin activities increased twofold in low-frequency-stimulated extensor digitorum longus (EDL) muscle, reaching a level intermediate between normal fast- and slow-twitch muscles. μ- and m-calpains were delineated by a calpain-specific zymographical assay that assessed total activities independent of calpastatin and distinguished between native and processed calpains. Contrary to normal EDL, structure-bound, namely myofibrillar and microsomal calpains, were abundant in soleus muscle. However, the fast-to-slow conversion of EDL was accompanied by an early translocation of cytosolic μ-calpain, suggesting that myofibrillar and microsomal μ-calpain was responsible for the twofold increase in activity and thus involved in controlled proteolysis during fiber transformation. This is in contrast to muscle regeneration where m-calpain translocation predominated. Taken together, we suggest that translocation is an important step in the control of calpain activity in skeletal muscle in vivo.
Differential regulation of tight junction permeability during development of the retinal pigment epithelium
Authors: Ban Yuriko , Rizzolo Lawrence J.
Published: December 2017

The retinal pigment epithelium (RPE) is an epithelial region of the blood-brain barrier. During embryogenesis, permeability of the barrier gradually decreases. A culture model of RPE development revealed differences in how tight junctions regulate the paracellular diffusion of ionic and nonionic solutes (Ban Y and Rizzolo LJ. Mol Vis 3: 18, 1997). To examine these differences, the permeation of ionic and nonionic monosaccharides was compared with mannitol, and the permeation of the alkali metals was compared with sodium. The order of permeation was 3- O-methlyglucose = glucosamine = mannitol > N-acetylneuraminic acid. The ratio of N-acetylneuraminic acid to mannitol permeability decreased with embryonic age of the RPE or exposure to retinal-conditioned medium. Neither the ratio nor the permeability was affected by inhibiting transcytosis. The ratio increased if tight junctions were disrupted in low-calcium medium. The permeation of cations followed the sequence cesium > rubidium > potassium = sodium > lithium and was unaffected by embryonic age or retinal-conditioned medium. These results are considered in terms of a model in which the size distribution, charge, or number of open junctional pores could be modulated. It suggests that different subpopulations of pores can be regulated independently during development.
Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization
Authors: Okamoto Curtis T. , Duman Joseph G. , Tyagarajan Kamala , McDonald Kent L. , Jeng Young Y. , McKinney Jeana , Forte Trudy M. , Forte John G.
Published: December 2017

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were α-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.
Functional and molecular characterization of an anion exchanger in airway serous epithelial cells
Authors: Loffing J. , Moyer B. D. , Reynolds D. , Shmukler B. E. , Alper S. L. , Stanton B. A.
Published: December 2017

Serous cells secrete Cl− and HCO3 − and play an important role in airway function. Recent studies suggest that a Cl−/HCO3 − anion exchanger (AE) may contribute to Cl− secretion by airway epithelial cells. However, the molecular identity, the cellular location, and the contribution of AEs to Cl− secretion in serous epithelial cells in tracheal submucosal glands are unknown. The goal of the present study was to determine the molecular identity, the cellular location, and the role of AEs in the function of serous epithelial cells. To this end, Calu-3 cells, a human airway cell line with a serous-cell phenotype, were studied by RT-PCR, immunoblot, and electrophysiological analysis to examine the role of AEs in Cl− secretion. In addition, the subcellular location of AE proteins was examined by immunofluorescence microscopy. Calu-3 cells expressed mRNA and protein for AE2 as determined by RT-PCR and Western blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat tracheal serous cells in situ. In Cl−/HCO3 −/Na+-containing media, the 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current ( I sc) was reduced by basolateral but not by apical application of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (50 μM) and 4,4′-dinitrostilbene-2,2′-disulfonic acid [DNDS (500 μM)], inhibitors of AEs. In the absence of Na+ in the bath solutions, to eliminate the contributions of the Na+/HCO3 − and Na+/K+/2Cl− cotransporters to I sc, CPT-cAMP stimulated a small DNDS-sensitive I sc. Taken together with previous studies, these observations suggest that a small component of cAMP-stimulated I sc across serous cells may be referable to Cl− secretion and that uptake of Cl− across the basolateral membrane may be mediated by AE2.
Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc
Authors: Ishihara Yukio , Sakurai Takashi , Kimura Taizou , Terakawa Susumu
Published: December 2017

The dynamic aspects of exocytosis, especially in the normal acinar tissue en bloc, have remained unclear. We visualized exocytosis directly in the tissue of the exocrine pancreas of rodents by video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol or cholecystokinin caused many of the zymogen granules in the apical pole to disappear abruptly. The exocytotic transients of individual granules were completed in 0.48–0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of ∼0.06 μm/s or less during stimulation. In the tissue preparation, granules located far from the apical pole frequently moved back and forth for 1–7 μm without showing exocytosis. Colchicine suppressed this movement and the late phase of the secretory response. Real-time (VEC-DIC) observation of granule dynamics revealed that the initial step of exocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.
15(S)-HETE modulates LTB4 production and neutrophil chemotaxis in chronic bronchitis
Authors: Profita Mirella , Sala Angelo , Riccobono Loredana , Pace Elisabetta , Paternò Alessandra , Zarini Simona , Siena Liboria , Mirabella Angela , Bonsignore Giovanni , Vignola Antonio M.
Published: December 2017

We evaluated the levels of 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of 15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 control and 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reverse phase high-performance liquid chromatography separation followed by specific RIA. 15-LO mRNA expression was determined by primed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than in control subjects. The percentage of cells expressing 15-LO mRNA was significantly higher in chronic bronchitis than in control subjects ( P < 0.01). Double staining for specific cell type markers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did not show evidence for 15-LO expression, suggesting that expression of 15-LO in neutrophils takes place on migration into the airways. Because 15(S)-HETE inversely correlated with the percentage of neutrophils in sputum of chronic bronchitis subjects, we studied the effect of 15(S)-HETE on leukotriene B4 (LTB4) production in vitro and evaluated the concentration of LTB4 in induced sputum and the contribution of LTB4 to the chemotactic activity of induced sputum samples ex vivo. The results obtained indicate that macrophages and neutrophils present within the airways of chronic bronchitis subjects express 15-LO mRNA; increased basal levels of 15(S)-HETE may contribute to modulate, through the inhibition of 5-lipoxygenase metabolites production, neutrophil infiltration and airway inflammation associated with chronic bronchitis.
Effects of dihydropyridine receptor II-III loop peptides on Ca2+ release in skinned skeletal muscle fibers
Authors: Lamb Graham D. , El-Hayek Roque , Ikemoto Noriaki , Stephenson D. George
Published: December 2017

In skeletal muscle fibers, the intracellular loop between domains II and III of the α1-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca2+ release channel in the sarcoplasmic reticulum. We examined the effects of synthetic peptide segments of this loop on Ca2+ release in mechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg2+ concentration ([Mg2+]; 1 mM), a 20-residue skeletal muscle DHPR peptide [AS(20); Thr671-Leu690; 30 μM], shown previously to induce Ca2+ release in a triad preparation, caused only small spontaneous force responses in ∼40% of fibers, although it potentiated responses to depolarization and caffeine in all fibers. The COOH-terminal half of AS(20)[AS(10)] induced much larger spontaneous responses but also caused substantial inhibition of Ca2+release to both depolarization and caffeine. Both peptides induced or potentiated Ca2+ release even when the voltage sensors were inactivated, indicating direct action on the Ca2+ release channels. The corresponding 20-residue cardiac DHPR peptide [AC(20); Thr793-Ala812] was ineffective, but its COOH-terminal half [AC(10)] had effects similar to AS(20). In the presence of lower [Mg2+] (0.2 mM), exposure to either AS(20) or AC(10) (30 μM) induced substantial Ca2+ release. Peptide CS (100 μM), a loop segment reported to inhibit Ca2+ release in triads, caused partial inhibition of depolarization-induced Ca2+ release. In toad fibers, each of the A peptides had effects similar to or greater than those in rat fibers. These findings suggest that the A and C regions of the skeletal DHPR II-III loop may have important roles in vivo.
Effect of astroglial cells on hypoxia-induced permeability in PBMEC cells
Authors: Fischer Silvia , Wobben Maria , Kleinstück Jan , Renz Dieter , Schaper Wolfgang
Published: December 2017

An in vitro model of the blood-brain barrier (BBB), consisting of porcine brain-derived microvascular endothelial cells (PBMEC), was used to evaluate the effect of astrocytes in the BBB disruption during hypoxia. Hypoxia-induced hyperpermeability was decreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, when glia cell-conditioned medium was used. Corresponding to effects on hypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor (VEGF) mRNA and protein expression, which recently was shown to be responsible for hypoxia-induced permeability changes in vitro. The effect on hypoxia-induced hyperpermeability and VEGF expression was specific for astroglia cells because conditioned medium from bovine smooth muscle cells (BSMC) did not show any effect. Immunocytochemistry revealed that 24 h of hypoxia disrupted the continuity of the tight junction protein, zonula occludens-1 (ZO-1), which lines the cytoplasmic face of intact tight junctions. These changes were prevented when hypoxia was performed in glia cell-conditioned medium. Results suggest that astrocytes protect the BBB from hypoxia-induced paracellular permeability changes by decreasing hypoxia-induced VEGF expression in microvascular endothelial cells.
Increased migration in late G1 phase in cultured smooth muscle cells
Authors: Fukui Ryosuke , Amakawa Masahiro , Hoshiga Masaaki , Shibata Nobuhiko , Kohbayashi Eiko , Seto Minoru , Sasaki Yasuharu , Ueno Teruo , Negoro Nobuyuki , Nakakoji Takahiro , Ii Masaaki , Nishiguchi Futoshi , Ishihara Tadashi , Ohsawa Nakaaki
Published: December 2017

Migration and proliferation of smooth muscle cells (SMC) contribute to neointimal formation after arterial injury. However, the relation between migration and proliferation in these cells is obscure. To discriminate between migration and proliferation, we employed a migration assay of SMC at different phases of the cell cycle. Serum-deprived SMC were synchronized in different phases of the cell cycle by addition of serum for various periods of time. Migration induced by platelet-derived growth factor B-chain homodimer was maximal in SMC that were predominantly in the late G1(G1b) phase. In addition, in nonsynchronized SMC, 65–75% of SMC that had migrated were in the G1bphase. Phosphorylated myosin light chain was enriched around the cell periphery in SMC in the G1b phase compared with SMC in the other cell cycle phases. Interestingly, the Triton X-100-insoluble fraction of myosin was remarkably decreased in G1b-enriched SMC. These findings suggest that migratory activity of SMC may be coupled with the G1b phase. The phosphorylation and retention of myosin might explain some of the properties responsible for increased migration.
Voltage dependence of l-arginine transport by hCAT-2A and hCAT-2B expressed in oocytes from Xenopus laevis
Authors: Nawrath Hermann , Wegener Jörg W. , Rupp Johanna , Habermeier Alice , Closs Ellen I.
Published: December 2017

Membrane potential and currents were investigated with the two-electrode voltage-clamp technique in Xenopus laevisoocytes expressing hCAT-2A or hCAT-2B, the splice variants of the human cationic amino acid transporter hCAT-2. Both hCAT-2A- and hCAT-2B-expressing oocytes exhibited a negative extracellularl-arginine concentration ([l-Arg]o)-sensitive membrane potential, additive to the K+ diffusion potential, when cells were incubated in Leibovitz medium (containing 1.45 mM l-Arg and 0.25 mM l-lysine). The two carrier proteins produced inward and outward currents, which were dependent on the l-Arg gradient and membrane potential. Ion substitution experiments showed that the hCAT-induced currents were independent of external Na+, K+, Ca2+, or Mg2+. The apparent Michaelis-Menten constant values at −60 mV, obtained from plots of l-Arg-induced currents against [l-Arg]o, were 0.97 and 0.13 mM in oocytes expressing hCAT-2A and hCAT-2B, respectively; maximal currents amounted to −194 ± 8 and −84 ± 2 nA, respectively. At saturating [l-Arg]o, the current-voltage relationships of hCAT-2A-expressing oocytes became steeper, yielding an additional conductance up to 2 μS/oocyte, whereas those of hCAT-2B-expressing oocytes were simply shifted to the right, resulting in voltage-independent difference currents. The distinct electrochemical properties of the two isoforms of hCAT-2 are assumed to contribute differentially to the membrane transport and the maintenance of cationic amino acids in various tissues.
Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression
Authors: Perez J. Xavier , Roig Teresa , Manzano Anna , Dalmau Mireia , Boada Jordi , Ventura Francesc , Rosa Jose L. , Bermudez Jordi , Bartrons Ramon
Published: December 2017

The ability to overexpress 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2)/(FBPase-2) or a truncated form of the enzyme with only the bisphosphatase domain allowed us to analyze the relative role of the kinase and the bisphosphatase activities in regulating fructose 2,6-bisphosphate (Fru-2,6-P2) concentration and to elucidate their differential metabolic impact in epithelial Mv1Lu cells. The effect of overexpressing PFK-2/FBPase-2 resulted in a small increase in the kinase activity and in the activity ratio of the bifunctional enzyme, increasing Fru-2,6-P2 levels, but these changes had no major effects on cell metabolism. In contrast, expression of the bisphosphatase domain increased the bisphosphatase activity, producing a significant decrease in Fru-2,6-P2concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well as a delay in cell cycle. These results provide support for Fru-2,6-P2 as a regulator of glycolytic flux and point out the role of glycolysis in cell cycle progression.