bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis
Authors: DiSanto Michael E. , Wang Ze , Menon Chandrakala , Zheng Yongmu , Chacko Thomas , Hypolite Joseph , Broderick Gregory , Wein Alan J. , Chacko Samuel
Published: December 2017

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ∼4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (∼10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ∼23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.
Intracellular signaling leads to the hypertrophic effect of neuropeptide Y
Authors: Goldberg Yaron , Taimor Gerhild , Piper Hans Michael , Schlüter Klaus-Dieter
Published: December 2017

Signal transduction pathways involved in the hypertrophic effect of neuropeptide Y (NPY) were investigated in adult cardiomyocytes. Reduction of transforming growth factor-β activity in serum-supplemented media abolished the induction of hypertrophic responsiveness to NPY. In responsive cells, NPY (100 nM) increased protein synthesis, determined as incorporation of [14C]phenylalanine, by 35 ± 15% ( P < 0.05, n = 16 cultures). In these cells, NPY activated pertussis toxin (PTx)-sensitive G proteins and phosphatidylinositol (PI) 3-kinase. PTx and inhibition of PI 3-kinase abolished the hypertrophic effect of NPY. NPY also activated protein kinase C (PKC) and mitogen-activated protein (MAP) kinase. Inhibition of these two kinases attenuated the induction of creatine kinase (CK)-BB but not the growth response to NPY. In conclusion, NPY stimulates protein synthesis in adult cardiomyocytes via activation of PTx-sensitive G proteins and PI 3-kinase and it induces the fetal-type CK-BB via activation of PKC and MAP kinase.
Regulation of angiotensin II-induced JAK2 tyrosine phosphorylation: roles of SHP-1 and SHP-2
Authors: Marrero Mario B. , Venema Virginia J. , Ju Hong , Eaton Douglas C. , Venema Richard C.
Published: December 2017

Angiotensin II (ANG II) exerts its effects on vascular smooth muscle cells through G protein-coupled AT1 receptors. ANG II stimulation activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway by inducing tyrosine phosphorylation, activation, and association of JAK2 with the receptor. Association appears to be required for JAK2 phosphorylation. In the present study, electroporation experiments with neutralizing anti-Src homology phosphatase-1 (SHP-1) and anti-SHP-2 antibodies and time course determinations of SHP-1 and SHP-2 activation and complexation with JAK2 suggest that the tyrosine phosphatases, SHP-1 and SHP-2, have opposite roles in ANG II-induced JAK2 phosphorylation. SHP-1 appears responsible for JAK2 dephosphorylation and termination of the ANG II-induced JAK/STAT cascade. SHP-2 appears to have an essential role in JAK2 phosphorylation and initiation of the ANG II-induced JAK/STAT cascade leading to cell proliferation. The motif in the AT1 receptor that is required for association with JAK2 is also required for association with SHP-2. Furthermore, SHP-2 is required for JAK2-receptor association. SHP-2 may thus play a role as an adaptor protein for JAK2 association with the receptor, thereby facilitating JAK2 phosphorylation and activation.
Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells
Authors: Park John M. , Borer Joseph G. , Freeman Michael R. , Peters Craig A.
Published: December 2017

Cultured rat bladder smooth muscle cells (SMC) were grown on collagen-coated silicone membranes and subjected to continuous cycles of stretch-relaxation. Semiquantitative RT-PCR analysis revealed a time-dependent increase in heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA levels after stretch, with maximal levels appearing after 4 h. Immunostaining for proHB-EGF revealed higher levels of HB-EGF protein in the stretched than in the nonstretched SMC. The ANG II receptor type 1 antagonist losartan markedly suppressed stretch-activated HB-EGF expression. ANG II levels were 3.3-fold higher in the stretch- than in the non-stretch-conditioned media. Stretch stimulation of bladder SMC that had been transiently transfected with an HB-EGF promoter-luciferase expression construct resulted in an 11-fold increase in reporter activity. Mechanical stretch induced a 4.7-fold increase in tritiated thymidine incorporation rate, and this was reduced by 25% in the presence of losartan. We conclude that mechanical stretch activates HB-EGF gene expression in bladder SMC and that this is mediated in part by autocrine ANG II secretion.
ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells
Authors: Hughes Bret A. , Takahira Masayuki
Published: December 2017

Inwardly rectifying K+ current ( I Kir) in freshly isolated bovine retinal pigment epithelial (RPE) cells was studied in the whole cell recording configuration of the patch-clamp technique. When cells were dialyzed with pipette solution containing no ATP, I Kir ran down completely in <10 min [half time ( t 1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained I Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM ATP was used, I Kir ran down by ∼71%. Mg2+ was a critical cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+( t 1/2 = 1.8 min). I Kir also ran down when the pipette solution contained 4 mM Mg2+ + 4 mM 5′-adenylylimidodiphosphate ( t 1/2 = 2.7 min) or 4 mM adenosine 5′- O-(3-thiotriphosphate) ( t 1/2 = 1.9 min), nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We conclude that the sustained activity of I Kirin bovine RPE requires intracellular MgATP and that the underlying mechanism may involve ATP hydrolysis.
Blockade by cAMP of native sodium channels of adult rat skeletal muscle fibers
Authors: Desaphy Jean-François , De Luca Annamaria , Camerino Diana Conte
Published: December 2017

Although the skeletal muscle sodium channel is a good substrate for cAMP-dependent protein kinase (PKA), no functional consequence was observed for this channel expressed in heterologous systems. Therefore, we investigated the effect of 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on the native sodium channels of freshly dissociated rat skeletal muscle fibers by means of the cell-attached patch-clamp technique. Externally applied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by ∼75% with no change in kinetics. Single-channel conductance and normalized activation curves were unchanged by CPT-cAMP. In contrast, steady-state inactivation curves showed a reduction of the maximal available current and a negative shift of the half-inactivation potential. Similar effects were observed with dibutyryl adenosine 3′,5′-cyclic monophosphate but not with cAMP, which does not easily permeate the cell membrane. Incubation of fibers for 1 h with 10 μM H-89, a PKA inhibitor, did not prevent the effect of CPT-cAMP. Finally, the β-adrenoreceptor agonist isoproterenol mimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM. These results indicate that cAMP inhibits native skeletal muscle sodium channels by acting within the fiber, independently of PKA activation.
Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation
Authors: Creer Michael H. , McHowat Jane
Published: December 2017

The present study was performed to characterize thrombin-stimulated phospholipase A2(PLA2) activity and the resultant release of lysophospholipids from endothelial cells. The majority of PLA2 activity in endothelial cells was membrane associated, Ca2+ independent, and arachidonate selective. Incubation with thrombin increased membrane-associated PLA2 activity using both plasmenylcholine and alkylacyl glycerophosphocholine substrates in the absence of Ca2+, with no increase in activity observed with phosphatidylcholine substrate. The increased PLA2 activity was accompanied by arachidonic acid and lysoplasmenylcholine (LPlasC) release from endothelial cells into the surrounding medium. Thrombin-induced changes were duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with the Ca2+-independent PLA2 inhibitor bromoenol lactone blocked thrombin-stimulated increases in PLA2 activity, arachidonic acid, and LPlasC release. Stimulation of protein kinase C (PKC) increased basal PLA2 activity and LPlasC production. Thrombin-stimulated PLA2 activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cells activates a PKC-activated, membrane-associated, Ca2+-independent PLA2 that selectively hydrolyzes arachidonylated, ether-linked phospholipid substrates, resulting in LPlasC and arachidonic acid release.
Characterization of CFTR expression and chloride channel activity in human endothelia
Authors: Tousson Albert , Van Tine Brian A. , Naren Anjaparavanda P. , Shaw George M. , Schwiebert Lisa M.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a low-conductance, cAMP-regulated chloride (Cl−) channel in a variety of cell types, such as exocrine epithelial cells. Our results demonstrate that human primary endothelial cells isolated from umbilical vein (HUVEC) and lung microvasculature (HLMVEC) also express CFTR as determined via RT-PCR and immunohistochemical and immunoprecipitation analyses. Moreover, Cl− efflux and whole cell patch-clamp analyses reveal that HUVEC ( n = 6 samples, P < 0.05) and HLMVEC ( n = 5 samples, P < 0.05) display cyclic nucleotide-stimulated Cl−transport that is inhibited by the CFTR selective Cl− channel blocker glibenclamide but not by the blocker DIDS, indicative of CFTR Cl− channel activity. Taken together, these findings demonstrate that human endothelial cells derived from multiple organ systems express CFTR and that CFTR functions as a cyclic nucleotide-regulated Cl− channel in human endothelia.
Colocalization of glycolytic enzyme activity and KATP channels in basolateral membrane of Necturusenterocytes
Authors: Dubinsky William P. , Mayorga-Wark O. , Schultz Stanley G.
Published: December 2017

86Rb fluxes through ATP-regulated K+(KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phospho enolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2,3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+pumps to KATP channels in the basolateral membrane, raise the possibility that “cross talk” between those channels and pumps (i.e., the “pump-leak parallelism”) may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.
Nitric oxide relaxes human myometrium by a cGMP-independent mechanism
Authors: Bradley Karri K. , Buxton Iain L. O. , Barber James E. , McGaw Terrence , Bradley Michael E.
Published: December 2017

The role of intracellular guanosine 3′,5′-cyclic monophosphate concentration ([cGMP]i) in nitric oxide (NO)-mediated relaxations in the uterus has become controversial. We found the NO donor S-nitroso-l-cysteine (CysNO) to potently (IC50 = 30 nM) inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased [cGMP]i significantly ( P < 0.001), and this increase was blocked by the guanylyl cyclase inhibitors methylene blue (10 μM) or LY-83583 (1 μM); however, pretreatment with these guanylyl cyclase inhibitors failed to block CysNO-mediated relaxations. Intracellular cAMP concentrations were not altered by treatment of tissues with 10 μM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or β-phenyl-1, N 2-etheno-cGMP did not significantly affect spontaneous contractility. Pretreatment of tissues with charybdotoxin [a calcium-dependent potassium channel (BK) blocker] completely reversed CysNO-induced relaxations. We conclude that NO is a potent inhibitor of spontaneous contractile activity in the nonpregnant human uterus and that, although guanylyl cyclase and BK activities are increased by NO, increases in [cGMP]i are not required for NO-induced relaxations in this tissue.
l-Arginine restores splenocyte functions after trauma and hemorrhage potentially by improving splenic blood flow
Authors: Angele Martin K. , Smail Nadia , Knöferl Markus W. , Ayala Alfred , Cioffi William G. , Chaudry Irshad H.
Published: December 2017

Several studies indicate that immune responses are markedly depressed early after onset of hemorrhage. Decreased organ blood flow has been implicated in the pathophysiology of altered immune responses after trauma-hemorrhage. In this regard, administration ofl-arginine has been shown to restore depressed intestinal and hepatic blood flow after trauma-hemorrhage, probably due to provision of substrate for constitutive nitric oxide synthase (cNOS). It remains unknown, however, whether administration ofl-arginine also ameliorates depressed splenic blood flow and whether this agent has any salutary effects on depressed splenocyte functions after trauma-hemorrhage. Male rats underwent sham operation or laparotomy and were bled to and maintained at a mean arterial blood pressure of 40 mmHg until 40% of maximum shed blood volume (MBV) was returned as Ringer lactate (RL). Hemorrhaged rats were then resuscitated with RL (4 times MBV over 1 h). During resuscitation, rats received 300 mg/kgl-arginine or saline (vehicle) intravenously; 4 h later, splenic blood flow, splenocyte proliferation, and splenocyte interleukin (IL)-2 and IL-3 were determined. Administration of l-arginine improved depressed splenic blood flow and restored depressed splenocyte functions after trauma-hemorrhage. Therefore, provision ofl-arginine during resuscitation after trauma-hemorrhage should be considered a novel and safe approach for improving splenic organ blood flow and depressed splenocyte functions under such conditions.
Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2
Authors: Denson Donald D. , Worrell Roger T. , Middleton Pamela , Eaton Douglas C.
Published: December 2017

To test the hypothesis that intracellular Ca2+activation of large-conductance Ca2+-activated K+ (BK) channels involves the cytosolic form of phospholipase A2(cPLA2), we first inhibited the expression of cPLA2 by treating GH3 cells with antisense oligonucleotides directed at the two possible translation start sites on cPLA2. Western blot analysis and a biochemical assay of cPLA2activity showed marked inhibition of the expression of cPLA2 in antisense-treated cells. We then examined the effects of intracellular Ca2+ concentration ([Ca2+]i) on single BK channels from these cells. Open channel probability ( P o) for the cells exposed to cPLA2 antisense oligonucleotides in 0.1 μM intracellular Ca2+ was significantly lower than in untreated or sense oligonucleotide-treated cells, but the voltage sensitivity did not change (measured as the slope of the P o-voltage relationship). In fact, a 1,000-fold increase in [Ca2+]ifrom 0.1 to 100 μM did not significantly increase P oin these cells, whereas BK channels from cells in the other treatment groups showed a normal P o-[Ca2+]iresponse. Finally, we examined the effect of exogenous arachidonic acid on the P oof BK channels from antisense-treated cells. Although arachidonic acid did significantly increase P o, it did so without restoring the [Ca2+]isensitivity observed in untreated cells. We conclude that although [Ca2+]idoes impart some basal activity to BK channels in GH3 cells, the steep P o-[Ca2+]irelationship that is characteristic of these channels involves cPLA2.
Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding
Authors: Zhang Jia-Zheng , Wu Yili , Williams Barbara Y. , Rodney George , Mandel Frederic , Strasburg Gale M. , Hamilton Susan L.
Published: December 2017

This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of125I-labeled CaM at both micromolar and nanomolar Ca2+concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.
AVP V1 receptor-mediated decrease in Cl− efflux and increase in dark cell number in choroid plexus epithelium
Authors: Johanson Conrad E. , Preston Jane E. , Chodobski Adam , Stopa Edward G. , Szmydynger-Chodobska Joanna , McMillan Paul N.
Published: December 2017

The cerebrospinal fluid (CSF)-generating choroid plexus (CP) has many V1 binding sites for arginine vasopressin (AVP). AVP decreases CSF formation rate and choroidal blood flow, but little is known about how AVP alters ion transport across the blood-CSF barrier. Adult rat lateral ventricle CP was loaded with36Cl−, exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of36Cl−. Effect of AVP at 10−12 to 10−7 M on the Cl− efflux rate coefficient (in s−1) was quantified. Maximal inhibition (by 20%) of Cl− extrusion at 10−9 M AVP was prevented by the V1 receptor antagonist [β-mercapto-β,β-cyclopentamethyleneproprionyl1, O-Me-Tyr2,Arg8]vasopressin. AVP also increased by more than twofold the number of dark and possibly dehydrated but otherwise morphologically normal choroid epithelial cells in adult CP. The V1 receptor antagonist prevented this AVP-induced increment in dark cell frequency. In infant rats (1 wk) with incomplete CSF secretory ability, 10−9 M AVP altered neither Cl− efflux nor dark cell frequency. The ability of AVP to elicit functional and structural changes in adult, but not infant, CP epithelium is discussed in regard to ion transport, CSF secretion, intracranial pressure, and hydrocephalus.
Estrogen modulates paracellular permeability of human endothelial cells by eNOS- and iNOS-related mechanisms
Authors: Cho Michael M. , Ziats Nicholas P. , Pal Dipika , Utian Wulf H. , Gorodeski George I.
Published: December 2017

Estradiol had a biphasic effect on permeability across cultures of human umbilical vein endothelial cells (HUVEC): at nanomolar concentrations it decreased the HUVEC culture permeability, but at micromolar concentrations it increased the permeability. The objective of the present study was to test the hypothesis that the changes in permeability were mediated by nitric oxide (NO)-related mechanisms. The results revealed dual modulation of endothelial paracellular permeability by estrogen. 1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related, Ca2+-dependent decrease in permeability was activated by nanomolar concentrations of estradiol, resulting in enhanced Cl−influx, increased cell size, and increases in the resistance of the lateral intercellular space ( R LIS) and in the resistance of the tight junctions ( R TJ); these effects appeared to be limited by the ability of cells to generate cGMP in response to NO. 2) An inducible NO synthase (iNOS)- and NO-related, Ca2+-independent increase in permeability was activated by micromolar concentrations of estradiol, resulting in enhanced Cl−efflux, decreased cell size, and decreased R LIS and R TJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems to the total paracellular resistance.
Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression
Authors: Troxell Megan L. , Chen Yih-Tai , Cobb Nicole , Nelson W. James , Marrs James A.
Published: December 2017

The role of E-cadherin, a calcium-dependent adhesion protein, in organizing and maintaining epithelial junctions was examined in detail by expressing a fusion protein (GP2-Cad1) composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin. All studies shown were also replicated using an analogous cell line that expresses a mutant cadherin construct (T151) under the control of tet repressor. Mutant cadherin was expressed at ∼10% of the endogenous E-cadherin level and had no apparent effect on tight junction function or on distributions of adherens junction, tight junction, or desmosomal marker proteins in established Madin-Darby canine kidney cell monolayers. However, GP2-Cad1 accelerated the disassembly of epithelial junctional complexes and delayed their reassembly in calcium switch experiments. Inducing expression of GP2-Cad1 to levels approximately threefold greater than endogenous E-cadherin expression levels in control cells resulted in a decrease in endogenous E-cadherin levels. This was due in part to increased protein turnover, indicating a cellular mechanism for sensing and controlling E-cadherin levels. Cadherin association with catenins is necessary for strong cadherin-mediated cell-cell adhesion. In cells expressing low levels of GP2-Cad1, protein levels and stoichiometry of the endogenous cadherin-catenin complex were unaffected. Thus effects of GP2-Cad1 on epithelial junctional complex assembly and stability were not due to competition with endogenous E-cadherin for catenin binding. Rather, we suggest that GP2-Cad1 interferes with the packing of endogenous cadherin-catenin complexes into higher-order structures in junctional complexes that results in junction destabilization.
Transport of fluid by lens epithelium
Authors: Fischbarg Jorge , Diecke Friedrich P. J. , Kuang Kunyan , Yu Bin , Kang Fengying , Iserovich Pavel , Li Yansui , Rosskothen Heinz , Koniarek Jan P.
Published: December 2017

We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the αTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37°C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in μl ⋅ h−1 ⋅ cm−2) 3.3 ± 0.3 for αTN4 cells ( n = 27) and 4.7 ± 1.0 for bovine layers ( n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 ± 0.62 μl ⋅ h−1 ⋅ lens−1( n = 5) and along the same pressure head at 12.5 ± 1.1 μl ⋅ h−1 ⋅ lens−1( n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.
Direct evidence of Na+/Ca2+exchange in squid rhabdomeric membranes
Authors: Bauer Paul J. , Schauf Heike , Schwarzer Andreas , Brown Joel E.
Published: December 2017

Na+/Ca2+exchange has been investigated in squid ( Loligo pealei) rhabdomeric membranes. Ca2+-containing vesicles have been prepared from purified rhabdomeric membranes by extrusion through polycarbonate filters of 1-μm pore size. After removal of external Ca2+, up to 90% of the entrapped Ca2+ could be specifically released by the addition of Na+; this finding indicates that most of the vesicles contained Na+/Ca2+exchanger. The Na+-induced Ca2+ efflux had a half-maximum value ( K 1/2) of ∼44 mM and a Hill coefficient of ∼1.7. The maximal Na+-induced Ca2+ efflux was ∼0.6 nmol Ca2+ ⋅ s−1 ⋅ mg protein−1. Similar Na+-induced Ca2+ effluxes were measured if K+ was replaced with Li+ or Cs+. Vesicles loaded with Ca2+ by Na+/Ca2+exchange also released this Ca2+by Na+/Ca2+exchange, suggesting that Na+/Ca2+exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of Ca2+ efflux enhanced by approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin, Na+/Ca2+exchange was characterized by a K 1/2 of ∼25 mM and a Hill coefficient of 1.6. For these vesicles, the maximal Na+-induced Ca2+ efflux was about twice as great as in control vesicles. We conclude that Na+/Ca2+exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors.
Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney
Authors: Beesley A. H. , Ortega B. , White S. J.
Published: December 2017

The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common “core exon” that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains.
Caffeine does not inhibit substance P-evoked intracellular Ca2+ mobilization in rat salivary acinar cells
Authors: Seo J. T. , Sugiya H. , Lee S. I. , Steward M. C. , Elliott A. C.
Published: December 2017

We used the Ca2+-sensitive fluorescent dye fura 2, together with measurements of intracellulard- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3], to assess the inhibitory effects of caffeine on signal transduction via G protein-coupled receptor pathways in isolated rat mandibular salivary acinar cells. ACh, norepinephrine (NE), and substance P (SP) all evoked substantial increases in the intracellular free Ca2+ concentration ([Ca2+]i). Responses to ACh and NE were markedly inhibited by prior application of 20 mM caffeine. The inhibitory effect of caffeine was not reproduced by phosphodiesterase inhibition with IBMX or addition of cell-permeant dibutyryl cAMP. In contrast to the ACh and NE responses, the [Ca2+]iresponse to SP was unaffected by caffeine. Despite this, SP and ACh appeared to mobilize Ca2+ from a common intracellular pool. Measurements of agonist-induced changes in Ins(1,4,5) P 3levels confirmed that caffeine inhibited the stimulus-response coupling pathway at a point before Ins(1,4,5) P 3generation. Caffeine did not, however, inhibit [Ca2+]iresponses evoked by direct activation of G proteins with 40 mM F−. These data show that caffeine inhibits G protein-coupled signal transduction in these cells at some element that is common to the muscarinic and α-adrenergic signaling pathways but is not shared by the SP signaling pathway. We suggest that this element might be a specific structural motif on the G protein-coupled muscarinic and α-adrenergic receptors.
Transferrin stimulates iron absorption, exocytosis, and secretion in cultured intestinal cells
Authors: Nuñez Marco T. , Tapia Victoria
Published: December 2017

The cellular mechanism by which basolateral transferrin (Tf) produces an increase in apical-to-basolateral Fe flux in Caco-2 cells was analyzed. After a pulse of 59Fe from the apical medium, three types of basolateral59Fe efflux were found: a59Fe efflux that was independent of the presence of Tf in the basolateral medium, a59Fe efflux in which59Fe left the cell bound to Tf, and a Tf-dependent 59Fe efflux in which 59Fe came off the cell not bound to Tf. Furthermore, addition of Tf to the basolateral medium doubled the exocytosis rate of Tf and increased the secretion of apolipoprotein A, a basolateral secretion marker. Both apotransferrin and Fe-containing Tf produced similar increases in59Fe efflux, Tf exocytosis, and apolipoprotein A secretion. The Ca2+ channel inhibitor SKF-96365 inhibited both the Tf-mediated increase in transepithelial Fe transport and the secretion of apolipoprotein A. Thus the activation of transepithelial Fe transport by Tf seems to be mediated by Ca2+ entry into the cells.
Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion
Authors: Mohan Sumathy , Mohan Natarajan , Valente Anthony J. , Sprague Eugene A.
Published: December 2017

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-κB (NF-κB). Here we investigate the hypothesis that low shear-induced activation of NF-κB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-l-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-κB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-κB activation. However, cytokine-induced NF-κB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-κB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.
Physiological significance of volume-regulatory transporters
Authors: O’Neill W. Charles
Published: December 2017

Research over the past 25 years has identified specific ion transporters and channels that are activated by acute changes in cell volume and that serve to restore steady-state volume. The mechanism by which cells sense changes in cell volume and activate the appropriate transporters remains a mystery, but recent studies are providing important clues. A curious aspect of volume regulation in mammalian cells is that it is often absent or incomplete in anisosmotic media, whereas complete volume regulation is observed with isosmotic shrinkage and swelling. The basis for this may lie in an important role of intracellular Cl− in controlling volume-regulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity but rather changes in the cellular content of osmotically active molecules. Volume-regulatory transporters are also closely linked to cell growth and metabolism, producing requisite changes in cell volume that may also signal subsequent growth and metabolic events. Thus, despite the relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology.
Molecular mechanisms of cytoadherence in malaria
Authors: Ho May , White Nicholas J.
Published: December 2017

Microbial pathogens subvert host adhesion molecules to disseminate or to enter host cells to promote their own survival. One such subversion is the cytoadherence of Plasmodium falciparum-infected erythrocytes (IRBC) to vascular endothelium, which protects the parasite from being removed by the spleen. The process results in microcirculatory obstruction and subsequent hypoxia, metabolic disturbances, and multiorgan failure, which are detrimental to the host. Understanding the molecular events involved in these adhesive interactions is therefore critical both in terms of pathogenesis and implications for therapeutic intervention. Under physiological flow conditions, cytoadherence occurs in a stepwise fashion through parasite ligands expressed on the surface of IRBC and the endothelial receptors CD36, intercellular adhesion molecule-1 (ICAM-1), P-selectin, and vascular adhesion molecule-1. Moreover, rolling on ICAM-1 and P-selectin increases subsequent adhesion to CD36, indicating that receptors can act synergistically. Cytoadherence may activate intracellular signaling pathways in both endothelial cells and IRBC, leading to gene expression of mediators such as cytokines, which could modify the outcome of the infection.
Regulation of total mitochondrial Ca2+ in perfused liver is independent of the permeability transition pore
Authors: Eriksson Ove , Pollesello Piero , Geimonen Erika
Published: December 2017

Triggering of the permeability transition pore (PTP) in isolated mitochondria causes release of matrix Ca2+, ions, and metabolites, and it has been proposed that the PTP mediates mitochondrial Ca2+ release in intact cells. To study the role of the PTP in mitochondrial energy metabolism, the mitochondrial content of Ca2+, Mg2+, ATP, and ADP was determined in hormonally stimulated rat livers perfused with cyclosporin A (CsA). Stimulation of livers perfused in the absence of CsA with glucagon and phenylephrine induced an extensive uptake of Ca2+, Mg2+, and ATP plus ADP by the mitochondria, followed by a release on omission of hormones. In the presence of CsA, the PTP was fully inhibited, but neither the hormone-induced uptake of Ca2+, ATP, or ADP by mitochondria nor their release after washout of hormones was significantly changed. We conclude that the regulation of sustained changes in mitochondrial Ca2+content induced by hormonal stimulation is independent of the PTP.
Alternate splicing in human Na+-MI cotransporter gene yields differentially regulated transport isoforms
Authors: Porcellati Francesca , Hosaka Yoshiyuki , Hlaing Tommy , Togawa Masaki , Larkin Dennis D. , Karihaloo Anil , Stevens Martin J. , Killen Paul D. , Greene Douglas A.
Published: December 2017

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+- myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent ∼1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3′-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3′ exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.
Prepulse-induced mode 2 gating behavior with and without β-adrenergic stimulation in cardiac L-type Ca channels
Authors: Hirano Yuji , Yoshinaga Takashi , Murata Mitsushige , Hiraoka Masayasu
Published: December 2017

Mode 2 gating of L-type Ca channels is characterized by high channel open probability ( NP o) and long openings. In cardiac myocytes, this mode is evoked physiologically in two apparently different circumstances: membrane depolarization (prepulse facilitation) and activation of protein kinase A. To examine whether the phosphorylation mechanism is involved during prepulse-induced facilitation of cardiac L-type Ca channels, we used isolated guinea pig ventricular myocytes to analyze depolarization-induced modal gating behavior under different basal levels of phosphorylation. In control, NP o measured at 0 mV was augmented as the duration of prepulse to +100 mV was prolonged from 50 to 400 ms. This was due to the induction of mode 2 gating behavior clustered at the beginning of test pulses. Analysis of open time distribution revealed that the prepulse evoked an extra component, the time constant of which is not dependent on prepulse duration. When isoproterenol (1 μM) was applied to keep Ca channels at an enhanced level of phosphorylation, basal NP o without prepulse was increased by a factor of 3.6 ± 2.2 ( n = 6). Under these conditions, prepulse further increased NP o by promoting long openings with the same kinetics of transition to mode 2 gating (τ ≅ 200 ms at +100 mV). Likewise, recovery from mode 2 gating, as estimated by the decay of averaged unitary current, was not affected after β-stimulation (τ ≅ 25 ms at 0 mV). The kinetic behavior independent from the basal level of phosphorylation or activity of cAMP-dependent protein kinase suggests that prepulse facilitation of the cardiac Ca channel involves a mechanism directly related to voltage-dependent conformational change rather than voltage-dependent phosphorylation.
KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells
Authors: Borok Zea , Mihyu Salim , Fernandes Valentino F. J. , Zhang Xiao-Ling , Kim Kwang-Jin , Lubman Richard L.
Published: December 2017

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2(normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance ( R t) and short-circuit current ( I sc) were determined before and after exposure. Na+ channel α-, β-, and γ-subunit and Na+-K+-ATPase α1- and β1-subunit mRNA levels were quantified by Northern analysis. Na+ pump α1- and β1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, I sc across AEC monolayers decreased by ∼60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel β-subunit mRNA expression was reduced by hyperoxia, whereas α- and γ-subunit mRNA expression was unchanged. Na+ pump α1-subunit mRNA was unchanged, whereas β1-subunit mRNA was decreased ∼80% by hyperoxia in parallel with a reduction in β1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by I sc) relative to normoxic controls. Levels of β1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.
Myofibroblasts. I. Paracrine cells important in health and disease
Authors: Powell D. W. , Mifflin R. C. , Valentich J. D. , Crowe S. E. , Saada J. I. , West A. B.
Published: December 2017

Myofibroblasts are a unique group of smooth-muscle-like fibroblasts that have a similar appearance and function regardless of their tissue of residence. Through the secretion of inflammatory and anti-inflammatory cytokines, chemokines, growth factors, both lipid and gaseous inflammatory mediators, as well as extracellular matrix proteins and proteases, they play an important role in organogenesis and oncogenesis, inflammation, repair, and fibrosis in most organs and tissues. Platelet-derived growth factor (PDGF) and stem cell factor are two secreted proteins responsible for differentiating myofibroblasts from embryological stem cells. These and other growth factors cause proliferation of myofibroblasts, and myofibroblast secretion of extracellular matrix (ECM) molecules and various cytokines and growth factors causes mobility, proliferation, and differentiation of epithelial or parenchymal cells. Repeated cycles of injury and repair lead to organ or tissue fibrosis through secretion of ECM by the myofibroblasts. Transforming growth factor-β and the PDGF family of growth factors are the key factors in the fibrotic response. Because of their ubiquitous presence in all tissues, myofibroblasts play important roles in various organ diseases and perhaps in multisystem diseases as well.
Protein kinase C inhibits Kv1.1 potassium channel function
Authors: Boland Linda M. , Jackson Katharine A.
Published: December 2017

The regulation by protein kinase C (PKC) of recombinant voltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC, caused persistent and large (up to 90%) inhibition of mouse, rat, and fly Shaker K currents. K current inhibition by PMA was blocked by inhibitors of PKC, and inhibition was not observed in control experiments with PMA analogs that do not activate PKC. However, site-directed substitution of potential PKC phosphorylation sites in the Kv1.1 protein did not prevent current inhibition by PMA. Kv1.1 current inhibition was also not accompanied by changes in macroscopic activation kinetics or in the conductance-voltage relationship. In Western blots, Kv1.1 membrane protein was not significantly reduced by PKC activation. The injection of oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibition of Kv1.1 currents. These data are consistent with the hypothesis that PKC-mediated inhibition of Kv1.1 channel function occurs by a novel mechanism that requires a C3 exoenzyme substrate but does not alter channel activation gating or promote internalization of the channel protein.
Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone
Authors: Cuthbert A. W. , Hickman M. E. , Thorn P. , MacVinish L. J.
Published: December 2017

1-Ethyl-2-benzimidazolone (EBIO) caused a sustained increase in electrogenic Cl− secretion in isolated mouse colon mucosae, an effect reduced by blocking basolateral K+ channels. The Ca2+-sensitive K+ channel blocker charybdotoxin (ChTX) and the cAMP-sensitive K+channel blocker 293B were more effective when the other had been added first, suggesting that both types of K+ channel were activated. EBIO did not cause Cl− secretion in cystic fibrosis (CF) colonic epithelia. In apically permeabilized colonic mucosae, EBIO increased the K+ current when a concentration gradient was imposed, an effect that was completely sensitive to ChTX. No current sensitive to trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethylchromane (293B) was found in this condition. However, the presence of basolateral cAMP-sensitive K+channels was demonstrated by the development of a 293B-sensitive K+ current after cAMP application in permeabilized mucosae. In isolated colonic crypts EBIO increased cAMP content but had no effect on intracellular Ca2+. It is concluded that EBIO stimulates Cl−secretion by activating Ca2+-sensitive and cAMP-sensitive K+ channels, thereby hyperpolarizing the apical membrane, which increases the electrical gradient for Cl− efflux through the CF transmembrane conductance regulator (CFTR). CFTR is also activated by the accumulation of cAMP as well as by direct activation.