bluuebunny/crossref_metadata_2025 huggingface dataset:
CD9 antigen mRNA is induced by hypertonicity in two renal epithelial cell lines
Authors: Sheikh-Hamad D. , Ferraris J. D. , Dragolovich J. , Preuss H. G. , Burg M. B. , Garcia-Perez A.
Published: December 2017

In diverse organisms, cells adapt to hyperosmotic stress by accumulating organic osmolytes. Mammalian renal medullary cells are routinely under osmotic stress. Two renal cell lines, Madin-Darby canine kidney (MDCK) and PAP-HT25, have been widely used to study mammalian osmotic regulation. In these epithelial cells, extracellular hypertonicity induces gene transcription of proteins directly involved in the metabolism and transport of organic osmolytes. This induction is relatively specific and not part of a generalized stress response. Little is known about the signal transduction pathway between cellular detection of extracellular osmolality and increased specific gene transcription. Here, using differential mRNA display polymerase chain reaction on MDCK cells in isotonic vs. hypertonic medium, we identify a cDNA product corresponding to CD9 antigen mRNA. CD9 antigen is a cell surface glycoprotein originally found in cells of the immune system. Although CD9 antigen has been structurally characterized, its function is unclear. We further demonstrate that CD9 antigen mRNA is present in MDCK and PAP-HT25 cells and that its mRNA abundance is induced by extracellular hypertonicity, but not by heat stress. Also, we show that accumulation of organic osmolytes markedly attenuates the CD9 mRNA induction, as only recently demonstrated with genes involved in the hyperosmotic stress response. This suggests a role for CD9 antigen in this response.
Alternate stimulation of apical CFTR by genistein in epithelia
Authors: Illek B. , Fischer H. , Machen T. E.
Published: December 2017

The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A. A cAMP-independent activation has been recently shown for the protein tyrosine kinase inhibitor genistein in CFTR-transfected NIH/3T3 fibroblasts. We further studied the role of genistein on Cl- secretion in HT-29/B6 and T84 colonic epithelial cells, which express native CFTR in their apical membranes. Transepithelial Cl- secretion was more effectively stimulated in T84 cells when compared with HT-29/B6 cells by mucosal perfusion with 50 microM genistein. Genistein, like the cAMP agonist forskolin, stimulated CFTR activity in cell-attached patches of single cells with similar slope conductances of 8.5 +/- 0.5 and 9.2 +/- 0.3 pS, respectively. Monolayers in Ussing chambers were basolaterally permeabilized with the pore former alpha-toxin, and gradient-driven Cl- current across the apical membrane (ICl) was measured. ICl was stimulated by serosal (i.e., cytosolic) cAMP (half-maximal stimulatory concentration = 9.8 +/- 1.9 microM). In the presence of cAMP (> 5 microM), subsequent mucosal, but not serosal, addition of genistein further increased Icl by approximately 16%; in the absence of cytosolic cAMP, genistein had no effect on ICl. The inactive analogue daidzein had no effect. When cAMP agonists were removed in the continued presence of genistein, ICl remained elevated in both permeabilized and intact monolayers as well as in cell-attached patches of single cells. In addition, genistein blocked K- currents across the basolateral membrane in apically amphotericin B-permeabilized monolayers (half maximal inhibitory concentration = 44.2 +/- 8.1 microM). Therefore, in intact epithelia, the overall secretory response to genistein is composed of stimulatory effects on the apical CFTR and inhibitory effects on the basolateral K+ conductance. We propose that genistein blocks a phosphatase, which regulates CFTR during cAMP-dependent stimulation.
Asymmetric voltage dependence of embryonic cardiac gap junction channels
Authors: Chen Y. H. , DeHaan R. L.
Published: December 2017

The voltage dependence of junctional conductance (Gj) and the unitary channel behavior of junctions in most pairs of 3-day, 7-day, and 18-day embryonic chick heart cells are symmetrical, i.e., they are independent of the direction of polarization of junctional potential (Vj). With either cell depolarized relative to its neighbor, unitary channel events have a maximal unit conductance (yj) near 240 pS and five substates at nearly equal 40-pS increments down to near 40 pS (6, 9). Using the dual patch-clamp technique, we demonstrate here that, in a fraction of such cell pairs, Vj-dependent channel kinetics are asymmetric. Depolarization of one cell causes a larger and faster voltage-dependent decline in Gj than the same depolarization of the other cell. In a typical asymmetric preparation, depolarization of the strongly Vj-dependent side caused an immediate series of 47 +/- 16 pS closing steps in single-channel current (ij), followed by virtual cessation of channel activity. After depolarization of the less Vj-sensitive side, channel activity (56 +/- 13 pS) continues for many seconds. The large-conductance states (160-240 pS) observed in the electrically symmetric junctions were absent from the asymmetric preparations. In these cell pairs, connexin (Cx) 42, Cx43, and Cx45 could be immunolocalized at the junctional surfaces. We postulate that the asymmetry of voltage dependence in some cell pairs results from a preponderance of heterochannels formed from these different connexins. The frequency of asymmetric pairs obtained from 3-day, 7-day, and 18-day embryonic hearts was 50% (4/8), 24% (6/25), and 12.5% (1/8), suggesting that the fraction of heterochannels in the junctions decreases with cardiac development.
Induction of a fast-oxidative phenotype by chronic muscle stimulation: mechanical and biochemical studies
Authors: Jarvis J. C. , Sutherland H. , Mayne C. N. , Gilroy S. J. , Salmons S.
Published: December 2017

We studied changes in the mechanical properties and myosin isoform composition of rabbit tibialis anterior muscles that were subjected to continuous stimulation at 2.5 Hz for up to 12 wk. The effects of stimulation at 2.5 Hz were less profound than those observed for the same duration of stimulation at 10 Hz (12). Stimulation at 10 Hz for 12 wk induced complete transformation to a slow-contracting muscle homogeneous in slow myosin isoforms; stimulation for the same period at 2.5 Hz resulted in moderate changes in contractile speed and a very small increase in the synthesis of slow myosin isoforms. On the other hand, the fatigue resistance of muscles stimulated at 2.5 Hz was as great, in both isometric and dynamic fatigue tests, as that of the muscles stimulated at 10 Hz. Thus entire fast skeletal muscles can be transformed to a state in which fast myosin isoforms continue to be synthesized, but the oxidative capacity is sufficient to support sustained working at a higher power output than that associated with slow muscle.
Chronic TPA treatment inhibits expression of proximal tubule-specific properties by LLC-PK1 cells
Authors: Amsler K. , Murray J. , Cruz R. , Chen J. L.
Published: December 2017

Confluent LLC-PK1 cell populations expressed progressively proximal tubule-specific properties, including gamma-glutamyl transpeptidase activity, sodium hexose symport activity, alkaline phosphatase activity, and villin protein. This was paralleled by an increase in villin protein manifested at the single cell level. Chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited expression of proximal tubule-specific properties at the levels of enzyme activity, protein content, and mRNA content. Inhibition occurred in all cells of the population. TPA treatment induced a decrease in total protein kinase C (PKC)-alpha protein content and a change in subcellular localization from predominantly soluble to predominantly particulate. PKC-epsilon protein content was unchanged by TPA treatment. PKC-epsilon was localized in both soluble and particulate fractions of control cells but was localized predominantly in particulate fractions after TPA treatment. PKC-delta was barely detectable in control cells, but content was markedly increased by TPA. These results suggest that TPA-induced inhibition of expression of proximal tubule-specific properties is mediated through modulation of content and/or subcellular localization of one or more PKC isozymes, likely PKC-alpha.
Differential long-term regulation of MEK and of p42 MAPK in rat glomerular mesangial cells
Authors: Schramek H. , Sorokin A. , Watson R. D. , Dunn M. J.
Published: December 2017

Constitutive stimulation of the mitogen-activated protein kinase (MAPK) activator MAPK/ERK kinase (MEK) is sufficient to promote long-term events such as cell differentiation, proliferation, and transformation. To evaluate a possible mechanism for the chronic regulation of MEK and p42 MAPK, we studied the long-term effects of fetal bovine serum (FBS), the G protein-coupled receptor agonist endothelin-1 (ET-1), and the protein tyrosine kinase-coupled receptor agonist platelet-derived growth factor BB (PDGF BB) on MEK and p42 MAPK in glomerular mesangial cells (GMC). FBS, ET-1, and PDGF BB led to a time-dependent increase in MEK-1 mRNA and protein expression without altering p42 MAPK mRNA and protein levels. FBS also induced MEK-1 mRNA expression in diverse cell types, including NIH/3T3 fibroblasts, A7r5 vascular smooth muscle cells, and Chinese hamster ovary cells. In GMC, cycloheximide inhibited MEK-1 mRNA induction but stimulated p42 MAPK mRNA expression in the absence and presence of FBS, ET-1, or PDGF. The FBS-induced increase in MEK-1 mRNA was accompanied by a sustained enhancement of MEK activity, as assessed by the ability of immunoprecipitated p45 MEK to activate recombinant p42 MAPK and hence phosphorylate myelin basic protein, and p42 MAPK activity. We conclude that, in GMC, MEK-1 acts like a delayed-early gene and that it can be chronically induced at the mRNA and protein level.
A model for the kinetic mechanism of sodium-coupled L-alanine transport in LLC-PK1 cells
Authors: Wilson J. J. , Randles J. , Kimmich G. A.
Published: December 2017

The kinetics of sodium-dependent L-alanine transport were characterized in ATP-depleted LLC-PK1 cells, which allows experimental imposition of an interior negative diffusion potential across the plasma membrane. Under these conditions a wide range of sodium concentrations can be studied without altering the membrane potential. When Na+ is the variable substrate, the apparent maximal velocity (V max) for transport changes nearly fourfold for the five different alanine concentrations studied (0.05-2.0 mM). In contrast, at five different sodium concentrations, ranging from 10 to 135 mM, the apparent V max with variable alanine remains nearly constant at 5.3 +/- 1.2 nmol.min-1.mg cell protein-1. The ratio of the two primary kinetic parameters, Michaelis constant (Km)/V max, varies markedly no matter which solute is treated as the variable illustrate. These data are consistent with a simultaneous ordered transport mechanism in which sodium binds before alanine to the transport protein at the extracellular surface of the membrane. Alanine-dependent 22Na+ influx is more than five times faster if unlabeled intracellular sodium is present than in its absence. Sodium-dependent influx of [14C]alanine is more rapid than net alanine flux only if unlabeled Na+ and alanine are both present intracellularly. These results indicate that the cotransporter can function more rapidly in an exchange mode than when it catalyzes net solute uptake and that Na+ is the first solute to be released at the intracellular side of the membrane. A model is presented that can be used for further quantitative analysis of the kinetic and functional properties of the cotransport system.
Swelling-activated anion conductance in skate hepatocytes: regulation by cell Cl- and ATP
Authors: Jackson P. S. , Churchwell K. , Ballatori N. , Boyer J. L. , Strange K.
Published: December 2017

Cell swelling activates an outwardly rectifying anion conductance in mammalian cells. The channel responsible for this conductance mediates volume-regulatory efflux of organic osmolytes such as taurine. We observed a similar conductance in hepatocytes from the skate Raja erinacea. Whole cell Cl- conductance was increased > 100-fold by a 2-fold increase in hepatocyte volume. The conductance was outwardly rectifying and had a relative cation permeability of approximately 0.2. Cation permeability was increased by reductions in patch pipette CsCl concentration, suggesting that the channel pore contains saturable anion and cation binding sites with different anion and cation affinities. The conductance had a broad anion selectivity and a relative taurine permeability of 0.17. Activation of the conductance required intracellular ATP or a nonhydrolyzable ATP analogue. Elevation of intracellular Cl- from 20 to 155 mM reduced current activation while the rate and extent of cell swelling were unaffected. Reduction of intracellular Cl- concentration to 5-10 mM caused spontaneous current activation without cell swelling. These results suggest that increases in cell Cl- levels increase the volume set point of the channel. We propose that the main function of the outwardly rectifying anion channel is nonselective transport of organic solutes.
Zinc transport in mammalian cells
Authors: Reyes J. G.
Published: December 2017

The importance of zinc in cell physiology is related mainly to its intracellular involvement in enzyme catalysis, protein structure, protein-protein interactions, and protein-oligonucleotide interactions. The mechanisms by which Zn2+ enters mammalian cells have been studied in a variety of cell systems. A review of this literature indicates that, in all cells, Zn2+ interacts with extracellular binding sites, which are likely to include binding sites involved in the subsequent translocation of this ion to the cell interior. Inside the cell, Zn2+ binds to cytosolic and organelle binding sites or is taken up by intracellular organelles. Despite these general conclusions, the mechanisms of the different transport and binding steps are, for most cell types, only partially solved. This review critically discusses the literature on mammalian Zn2+ transport and outlines some critical points for future research of the mechanisms of transport of this ion.
Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel. However, very little is known about the endogenous mechanism(s) of deactivation of CFTR-Cl conductance (CFTR-GCl) in vivo. We studied the action of endogenous phosphatases in regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)- and ATP-induced CFTR-GCl in the apical membrane of microperfused preparations of basolaterally permeabilized native sweat duct. Activation of CFTR-GCl was monitored by measuring the apical Cl diffusion potentials and GCl, which spontaneously deactivated on removal of cAMP. This spontaneous loss of CFTR-GCl activity could be prevented by a cocktail of phosphatase inhibitors (fluoride, vanadate, and okadaic acid). We studied the effects of each of these phosphatase antagonists on the rate of deactivation of CFTR-GCl after cAMP washout. In contrast to vanadate or fluoride, okadaic acid virtually prevented deactivation of CFTR-GCl after cAMP washout. We conclude that either or both protein phosphatases 1 and 2A are responsible for the dephosphorylation deactivation of CFTR-GCl in vivo.
Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells
Authors: Lynch R. M. , Carrington W. , Fogarty K. E. , Fay F. S.
Published: December 2017

Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.
Caffeine-induced chloride current in dissociated rat hepatocytes
Authors: Yamashita Y. , Akaike N.
Published: December 2017

Current responses to caffeine in single hepatocytes dissociated from adult rat liver were investigated with the conventional whole cell patch-recording configuration. Caffeine produced a sustained inward current (Icaf) with increasing conductance at a holding potential of -40 mV. The reversal potential of Icaf was close to the Cl- equilibrium potential. Icaf was not affected by the internal perfusion of 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or Cs+, whereas the Ca(2+)-activated K+ outward current elicited by A-23187 was inhibited by intracellular BAPTA or Cs+. A 1 mM 3-isobutyl-1-methylxanthine (IBMX) was about equipotent to 1 mM caffeine in inducing the current. Icaf was not modulated by the external application of N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), a cyclic nucleotide-dependent protein kinase inhibitor, or intracellular perfusion with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). It was concluded that caffeine induced an increase in membrane Cl- conductance without utilizing the rise of intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate (cAMP) and without mediating G protein, suggesting the possible existence of caffeine receptor-Cl- channel complexes on liver plasma membrane.
Palmitoyl-CoA potentiates the Ca2+ release elicited by cyclic ADP-ribose
Authors: Chini E. N. , Dousa T. P.
Published: December 2017

Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs that ultimately activates the ryanodine channel. We now report that certain long-chain acyl-CoA derivative metabolites (14-18 carbons in length), such as palmitoyl-CoA, greatly potentiate the effect of cADPR on Ca2+ release. Furthermore, in higher concentrations, palmitoyl-CoA and other closely related long-chain acyl-CoA derivatives trigger Ca2+ release apparently through the ryanodine channel in sea urchin egg homogenates. Palmitoyl-CoA-induced Ca2+ release was suppressed by ruthenium red, spermine, and the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide, which all prevent activation of the ryanodine channel, but not by heparin or thionicotinamide-NADP. In addition, cADPR was able to desensitize the sea urchin egg homogenates to the subsequent Ca2+ release induced by palmitoyl-CoA and vice versa. In contrast, neither inositol 1,4,5-trisphosphate (IP3) nor the newly identified Ca2+ release agonist nicotinate adenine dinucleotide phosphate was able to desensitize the homogenate to palmitoyl-CoA, indicating that palmitoyl-CoA probably acts selectively by activating the ryanodine channel, but, unlike cADPR, palmitoyl-CoA might act directly on this channel. Finally, we found that palmitoyl-CoA was able to counteract the inhibitory effect of Mg2+ and spermine, which, in physiological concentrations, suppress specifically the cADPR-induced Ca2+ release. We propose that palmitoyl-CoA, present in micromolar concentrations, may trigger Ca2+ release through the ryanodine channel and, in lower concentrations, may increase the sensitivity of the Ca2+ release system to cADPR. Thus palmitoyl-CoA may serve as a regulatory link between the intermediary metabolism and the cADPR-induced Ca2+ release signaling pathway.
Cellular ATP release by the cystic fibrosis transmembrane conductance regulator
Authors: Prat A. G. , Reisin I. L. , Ausiello D. A. , Cantiello H. F.
Published: December 2017

Recent studies from our laboratory indicate that members of the ATP-binding cassette (ABC) family of transporters, including P-glycoprotein and cystic fibrosis transmembrane conductance regulator (CFTR), are ATP-permeable channels. The physiological relevance of this novel transport mechanism is largely unknown. In the present study, intra- and extracellular ATP content, cellular ATP release, and O2 consumption before and after adenosine 3',5'-cyclic monophosphate (cAMP) stimulation were determined to assess the role of CFTR in the transport of ATP under physiological conditions. The functional expression of CFTR by the stable transfection of mouse mammary carcinoma cells, C1271, with human epithelial CFTR cDNA resulted in a stimulated metabolism, since both basal and cAMP-inducible O2 consumption were increased compared with mock-transfected cells. The stimulated (but not basal) O2 consumption was inhibited by diphenyl-2-carboxylic acid (DPC), a known inhibitor of CFTR. CFTR expression was also associated with the cAMP-activated and DPC-inhibitable release of intracellular ATP. The recovery of intracellular ATP from complete depletion after metabolic poisoning was also assessed under basal and cAMP-stimulated conditions. The various maneuvers indicate that CFTR may be an important contributor to the release of cellular ATP, which may help modify signal transduction pathways associated with secretory Cl- movement or other related processes. Changes in the CFTR-mediated delivery of nucleotides to the extracellular compartment may play an important role in the onset and reversal of the cystic fibrosis phenotype.
Renal Na+/H+ exchanger isoforms and their regulation by thyroid hormone
Authors: Azuma K. K. , Balkovetz D. F. , Magyar C. E. , Lescale-Matys L. , Zhang Y. , Chambrey R. , Warnock D. G. , McDonough A. A.
Published: December 2017

Na+ crosses the luminal membrane of the proximal tubule primarily via Na+/H+ exchange (NHE), and NHE activity is influenced by thyroid status. Pharmacological, immunological, and kinetic studies indicate multiple isoforms of NHE, and four full-length cDNAs have been cloned to date. The aims of this study were to determine which NHE mRNAs (NHE1, -2, -3, and -4) were expressed in the rat proximal tubule, the relative abundance of each in the renal cortex, and the effect of thyroid status on their expression. By blot hybridization of poly(A)+ RNA, all NHE isoform mRNAs were detected in the rat renal cortex; NHE1, -2, and -3 in the proximal tubule; and NHE1 and -3 in LLC-PK1 cells. NHE3 mRNA abundance was fourfold higher than the other three isoforms in renal cortex. The effect of thyroid status was assessed in renal cortex from euthyroid, hypothyroid, and hyperthyroid rats. Although none of the NHE mRNA levels was altered in the transition from euthyroid to hypothyroid states, both NHE2 and NHE3 mRNA levels increased 1.5-fold in the transition from hypo- to hyperthyroidism. NHE3 protein, measured by immunoblot with the use of an NHE3-specific antibody, was detected at 83-85 kDa in renal cortex and codistributed on sorbitol gradients with the brush-border marker alkaline phosphatase. No significant difference in NHE3 protein abundance was detected between hypothyroid and hyperthyroid rats. In conclusion, in the renal cortex, the NHE3 isoform predominates at the mRNA level, is expressed in apical membranes, and increases at the mRNA but not the protein levels in response to thyroid hormone treatment, suggesting parallel changes in synthesis and turnover of NHE3 by thyroid hormone.
Calcium chelators enhance 45Ca accumulation in permeablized synaptosomes and in microsomes
Authors: Moore J. E. , Abercrombie R. F.
Published: December 2017

The study of intracellular Ca2+ regulation usually requires using calcium chelators to adjust [Ca2+]. We examined the effects of these chelators on calcium accumulation in microsomes and saponin-permeabilized synaptosomes to assess their influence on apparent transport properties. At a fixed free Ca2+ of 0.6 microM, increasing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) and total Ca2+ enhanced ATP-dependent 45Ca sequestration in synaptosomes and microsomes. The EGTA-Ca complex did not change the maximal initial calcium uptake rate or maximal steady-state accumulation. Rather, EGTA/Ca increased the apparent affinity of the microsomal transporter for Ca2+. The presence of the organic anion transport inhibitor probenicid (2.5 mM) had no effect on 45Ca accumulation in the presence of EGTA. Replacing part of the Ca2+ with Ni2+ but maintaining [Ca2+] approximately constant reduced 45Ca uptake, suggesting that the Ni-EGTA complex did not stimulate 45Ca transport. Our results imply that EGTA is not actively transported across the endoplasmic reticulum membrane, nor does the divalent ion-bound form of EGTA change the properties of the transporter. EGTA, and other mobile calcium chelators with similar structures, e.g., 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, indo 1, and fluo 3, may increase calcium uptake by delivering more Ca2+ to its transport site.
Sphingolipid actions on sodium and calcium currents of rat ventricular myocytes
Authors: Yasui K. , Palade P.
Published: December 2017

Sphingosine, an endogenous phospholipid known to produce significant decreases in myoplasmic Ca2+ transients, was shown to have a pronounced inhibitory effect on inward Na+ and L-type Ca2+ currents in rat ventricular myocytes. Sphingosine action was accompanied by a slowing of inactivation of both kinds of current. Both sphingosine and sphingosylphosphorylcholine (SPC) caused depolarizing shifts in the activation curves for the two channels. In tests on Ca2+ currents, sphingosine neither showed high affinity for inactivated states nor exhibited any use dependence. The mechanism of the blocking action of sphingosine does not appear to involve effects on bulk surface charge or, at least for Ca2+ channels, a voltage-dependent block. Instead, the results appear most consistent with an effect of sphingosine on channel gating. The shift in the voltage dependence of channel activation by sphingosine and SPC appears likely to be a feature of both the hydrocarbon chains and the net positive charge of these amphiphiles.
Immunolocalization of the mNav2.3 Na+ channel in mouse heart: upregulation in myometrium during pregnancy
Authors: Knittle T. J. , Doyle K. L. , Tamkun M. M.
Published: December 2017

mNav2.3 is a putative voltage-dependent sodium channel (NaC) gene expressed in both mouse heart and uterus that shares only 45% amino acid identity with NaCs from gene subfamily 1. Immunofluorescence studies using polyclonal antibodies against two distinct epitopes revealed that mNav2.3 protein in heart colocalized with nerve-specific antibody binding. Similar mNav2.3-specific antibody staining was observed in virgin uterus. However, mNav2.3 expression in uterine nerve disappeared during late pregnancy, concurrent with an appearance in both the longitudinal and circular uterine smooth muscle, which reached a maximum at term and quickly declined within 2 days postpartum. mNav2.3 expression in term uterus often colocalized on the myocyte surface with connexin 43. The immunofluorescence results are supported by Western analysis in which the 217-kDa NaC increased during late pregnancy and declined 2 days postpartum. These data provide perhaps the most dramatic example of NaC regulation. The acute and transient upregulation in myometrium during gestation suggests the Nav2.3 channel plays a role in uterine function at term.
Mechanical measurements from isolated cardiac myocytes using a pipette attachment system
Authors: Palmer R. E. , Brady A. J. , Roos K. P.
Published: December 2017

Single rat left ventricular myocytes were attached at both ends using a newly described double-barreled micropipette technique. This attachment procedure enabled the measurement of the active and passive mechanical properties of chemically skinned cells that showed little structural deformation. The force and oscillatory stiffness (100 Hz) of the cells were measured with a high signal-to-noise ratio, and the sarcomere length throughout the entire cell was monitored using image analysis. The passive properties were investigated from the resting sarcomere length to > 3 microns. Analysis of the sarcomere behavior indicated a high level of homogeneity throughout the cell. The attachment method supported the full activation of the cells by increased free Ca2+ (pCa 4.5), which produced 22.3 mN/mm (mean sarcomere length 2.11 microns). A force/pCa relationship was determined, which, when fitted according to the Hill equation, gave parameters of nH = 2.62 and pCa50 = 5.58. The described techniques allow the accurate study of the mechanical properties of single myocytes with increased fidelity and reliability over the preexisting methods.
Differential regulation of Na+-K+-ATPase gene expression by corticosteriods in vascular smooth muscle cells
Authors: Muto S. , Nemoto J. , Ohtaka A. , Watanabe Y. , Yamaki M. , Kawakami K. , Nagano K. , Asano Y.
Published: December 2017

To determine whether gluco- and mineralocorticoids have specific actions on Na+-K+-ATPase gene expression in vascular tissue, we used Northern blot analysis to compare the effects of dexamethasone (Dex) and aldosterone (Aldo) on Na+-K+-ATPase alpha1 and beta1-subunit mRNA expression in cultured vascular smooth muscle cells from rat aortae. Dex at 10(-6)M increased alpha1 -mRNA level 2.5-fold at 24 h and beta1-mRNA level 9.9-fold at 12 h. Aldo at 10(-6)M increased alpha1-mRNA 2.7-fold at 48 h and beta1-mRNA level 10.9-fold at 6 h. The half-maximal stimulation of both alpha1 and beta1-mRNA levels occurred at a concentration of 5-7 X 10(-9)M Dex, whereas it occurred at a concentration of 2-3 X 10(-9)M Aldo. The glucocorticoid receptor antagonist RU-38486 inhibited both Dex- and Aldo-mediated induction of beta1-mRNA. The mineralocorticoid receptor antagonist spironolactone inhibited Aldo-mediated induction of beta1-mRNA, whereas it had no effect on Dex-mediated induction of beta1-mRNA. Removal of Na+ from the extracellular medium (isosmotic replacement with choline) caused no effect on Dex-mediated induction beta1-mRNA, whereas it inhibited Aldo-mediated induction of beta1-mRNA. Addition of a specific inhibitor of the Na+/H+ exchange, ethylisopropylamiloride, had no effect on Dex-mediated induction of beta1-mRNA, whereas it resulted in a significant inhibition of Aldo-mediated induction of beta1-mRNA. We conclude that 1) both Dex and Aldo induce Na+-K+-ATPase alpha1- and beta1-mRNA expression in a time- and dose-dependent manner; 2) Dex-mediated induction of beta1-mRNA occurs only through glucocorticoid receptors, whereas Aldo-mediated induction of beta1-mRNA occurs through both gluco- and mineralocorticoid receptors; and 3) Dex-mediated induction of beta1-mRNA occurs through Na+-independent mechanisms, whereas Aldo-mediated induction of beta1-mRNA, at least in part, occurs through Na+-dependent mechanisms, including stimulation of the Na+/H+ exchange.
Xenopus oocytes express multiple receptors for LPA-like lipid mediators
Authors: Liliom K. , Murakami-Murofushi K. , Kobayashi S. , Murofushi H. , Tigyi G.
Published: December 2017

In Xenopus laevis oocytes, both lysophosphatidic acid (LPA) and a cyclic phosphate-containing analogue 1-acyl-sn-glycero-2,3-cyclic phosphate (cLPA) isolated from Physarum polycephalum activated oscillatory Cl- currents. cLPA elicited oscillatory currents only when applied extracellularly and, similarly to LPA, evoked homologous desensitization. cLPA applied to oocytes previously desensitized b y LPA failed to elicit a current, indicating that LPA completely desensitized the cLPA receptors. In contrast, when oocytes were desensitized by cLPA, LPA still evoked large currents. The lack of heterologous desensitization between cLPA and LPA indicates that the former acts on a distinct receptor subpopulation(s), which is also activated by LPA. The alkyl-ether analogue 1-hexadecyl-2-lyso-sn-glycero-3-phosphate (16:0-GP) and dioleoyl-phosphatidic acid (18:1-PA) showed heterologous desensitization patterns similar to that of LPA with regard to cLPA. Complete heterologous desensitization was obtained between LPA and 16:0-GP or 18:1-PA. These observations demonstrate the simultaneous expression of at least two different types of receptors for LPA-like lipid mediators on Xenopus oocytes and that these receptors show different pharmacological properties in their selectivity to cLPA.
Coordinate modulation of glucocorticoid receptor and glutaminase gene expression in LLC-PK1-F+ cells
Authors: Gowda B. , Sar M. , Mu X. , Cidlowski J. , Welbourne T.
Published: December 2017

The effect of glucocorticoid receptor on glutaminase gene expression and related glutamine metabolism was studied in proximal tubule-like LCC-PK1-F+ cells. These cells express functional glucocorticoid receptors as demonstrated by immunoreactivity with antiglucocorticoid receptor antibody, specific ligand binding, and a 14-fold increase in chloramphenicol acetyltransferase (CAT) reporter gene activity after exposure to dexamethasone (10(-6)M). Dexamethasone exposure for 18 h increased glutaminase mRNA and activity by 32 and 42%, respectively (both P< 0.05, paired t-test), associated with a small (9%) but significant increase in glutamine utilization (P<0.05). In an effort to elicit a greater response, endogenous glucocorticoid receptors were supplemented by transfecting cells with a plasmid, pMAMGR, expressing the rat glucocorticoid receptor gene. Transfected cells expressed a 39-fold increase in CAT activity with dexamethasone treatment, confirming a higher level of functional receptors, but glutaminase mRNA and activity were now decreased by 34 and 32%, respectively, associated with a 15% fall in glutamine utilization after 18-h exposure to dexamethasone. This biphasic response in glutaminase gene expression was mirrored by glucocorticoid receptor mRNA that increased 41% after dexamethasone in LLC-PK(1)-F+ cells, but decreased 63% after transfection (both P<0.05). These findings are consonant with glucocorticoid receptor gene modulation of glutaminase gene expression and glutamine utilization.
Production and localization of cGMP and PGE2 in nitroprusside-stimulated rat colonic ion transport
Authors: Wilson K. T. , Vaandrager A. B. , De Vente J. , Musch M. W. , De Jonge H. R. , Chang E. B.
Published: December 2017

Nitrovasodilators, such as sodium nitroprusside (SNP), release nitric oxide (NO) and stimulate intestinal electrolyte transport. However, the second messengers involved in this process are unknown. NO stimulates soluble guanylate cyclase activity in other tissues, but stimulation of this enzyme has not previously been described for intestine. We report a 20-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) production by radioimmunoassay in colonic mucosal strips stimulated with SNP. SNP also caused a significant increase in prostaglandin (PG) E2 release but did not stimulate release of the prostanoids thromboxane B2 or 6-keto-PGF1alpha. Stimulation of isolated colonic crypts and the remaining subepithelial mucosa demonstrated that the latter was the major source of the increases in cGMP and PGE2. Immunostaining of colonic mucosa revealed minimal basal cGMP immunoreactivity but large increases in abundance, localizing to the subepithelium, after SNP treatment. Under basal conditions, there was diffuse immunostaining for constitutive NO synthase in both the epithelial and subepithelial compartments, which was corroborated with NADPH diaphorase staining. In conclusion, SNP was an NO donor stimulates production of cGMP and PGE2 from the subepithelium. NO may be an important mediator of colonic secretion and other processes predominantly via its direct effects on cells of the lamina propria.
EGF modulation of Na+/H+ antiport in rat hepatocytes: different sensitivity in adult and fetal cells
Authors: Incerpi S. , Spagnuolo S. , Terenzi F. , Leoni S.
Published: December 2017

The modulation by epidermal growth factor (EGF) of the Na+/H+ antiport in fetal and adult rat hepatocytes was studied in nominally HCO3- free solution. EGF (10 nM) activated the antiport in adult rat hepatocytes by 0.22 +/- 0.03 (mean +/- SD;n=10) pH units over basal value, measured with the fluorescent pH-sensitive intracellular probe, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of EGF was inhibited by amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA), by ouabain, inhibitor of the Na+ pump, and by erbstatin analogue, an inhibitor of the tyrosine kinase activity of the EGF receptor. The effect of EGF on Na+/H+ antiport in adult rat hepatocytes appeared to be mediated by both protein kinase C (PKC) and G protein system. No effect of EGF and phorbol 12-myristate 13-acetate, an activator of PKC, on the Na+/H+ antiport was observed in fetal hepatocytes of 20 and 22 days. A different sensitivity of the antiport to high concentrations of amiloride and EIPA suggests that altered amount of the Na+/H+ antiport units or different isoforms could be expressed in fetal compared with adult cells.
BAY K 8644 depresses excitation-contraction coupling in cardiac muscle
Authors: McCall E. , Bers D. M.
Published: December 2017

We determined the effect of the dihydropyridine L-type Ca channel agonist BAY K 8644 (BAY) on excitation-contraction (E-C) coupling in isolated ferret ventricular myocytes using whole cell voltage clamp. The sarcoplasmic reticulum (SR) Ca load during the test pulses, assessed by caffeine-induced contractures, was similar in the presence and absence of BAY, with extracellular Ca concentration lowered from 3 to 1 mM in BAY. The relationship between L-type Ca current (ICa) and contraction was assessed, with current and contractions measured during depolarizations from -40 to between -30 and +50 mV after a conditioning train (to ensure constant SR Ca load). BAY shifted the current-contraction relationship downward, such that, for a given ICa and SR Ca load, the contraction elicited was much smaller in the presence of BAY. BAY also induced a characteristic negative shift in the the current-voltage relationship. We conclude that BAY decreases the efficacy of a given Ca current to induce SR Ca release during E-C coupling in ferret cardiac tissue (in contrast to the BAY-induced increase of resting SR Ca release). This may reflect an alteration in the state of the SR Ca release channel due to BAY binding to dihydropyridine receptors.
Response of LLC-PK1-F+ cells to metabolic acidosis
Authors: Mu X. , Welbourne T.
Published: December 2017

The role of extracellular glutamate formation as opposed to cellular glutamate removal in regulating monolayer glutamate content in response to metabolic acidosis was studied in LLC-PK1-F+ cells. Exposure to metabolic acidosis (14 mM bicarbonate; pH 7.1) for 18 h resulted in 24% fall in monolayer glutamate content. Of this, approximately one-half could be attributed to enhanced glutamate removal via glutamate dehydrogenase, consistent with a rise in ammonium production. The remainder appears due to reduced extracellular glutamate formation as a consequence of diminished gamma-glutamyltranspeptidase (gamma-Gt) activity. Metabolic acidosis, but not respiratory acidosis, resulted in a 33% fall in gamma-Gt activity and a proportional fall in extracellular glutamate formation; glutamate transport into these cells was not rate limiting in acidosis. Overall glutamine utilization decreased 36%, reflecting the fall in gamma-Gt activity as well as a decrease in a pH-sensitive glutamine uptake, whereas glutamine transport coupled to the phosphate-dependent glutaminase flux increased. It is noteworthy that the increased ammonium produced in metabolic acidosis was preferentially secreted into the apical compartment; acid secretion, but not production, was similarly increased. Thus reduced cellular glutamate appears to coordinate activation of intracellular glutaminase to the apical membrane exchanger, consistent with the functioning kidney response to metabolic acidosis.
Electrophysiological characterization of RACTK1 K+ channel in stable cell line
Authors: Suzuki M. , Murata M. , Ikeda M. , Miyoshi T. , Imai M.
Published: December 2017

RACTK1 is a pH-sensitive K+ channel cloned from rabbit renal collecting tubule cells. To characterize the function of this K+ channel in more detail, RACTK1 was transfected to an established cell line and the patch-clamp study was performed. cDNA of RACTK1 was inserted in the pMAM vector and transfected to Chinese hamster ovary cells. In one of 36 cell lines, the channel protein as expressed by the dexamethasone-induced mRNA and was detected by the specific antibody. The RACTK1 K+ channel with 80 pS was consistently observed. In inside-out patch, Ca2+ at concentrations higher than 500 nM activated the channel. Open probability was decreased by protein kinase A (from 45 to 4.2%, n+6) but not by protein kinase C. Whole cell currents of the transformed cells represented K+ conductance that was not blocked by an addition of charybdotoxin but by apamin. RACTK1 K+ channel has similar, though not identical, characteristics to the Ca2+ -activated K+ channel. RACTK1 might therefore encode a subunit of the intermediate conductance Ca2+ -activated K+ channel observed in the apical membrane of the rabbit renal collecting duct.
IMP reamination to AMP in rat skeletal muscle fiber types
Authors: Tullson P. C. , Arabadjis P. G. , Rundell K. W. , Terjung R. L.
Published: December 2017

Inosine 5'-monophosphate (IMP) reamination in skeletal muscle fiber sections of the rat hindlimb was studied. High IMP concentrations were established during ischemic contractions in each fiber section: 3.1, 2.8, or 0.6 mumol/g in the fast-twitch white (FTW), fast-twitch red (FTR), and slow-twitch red (STR) muscle sections, respectively. Thereafter blood flow was restored and stimulation was discontinued to allow reamination of IMP. After 0, 2, 5, 10, 15, or 20 min of recovery, muscle sections were freeze-clamped and analyzed for metabolite contents. IMP was nearly fully reaminated after 10 and 20 min of recovery in STR and FTR muscles, respectively. Reamination in TW fibers was delayed and slower, with only 50% of the IMP reaminated after 20 min of recovery. Significant recovery (approximately 75%) of phosphocreatine occurs in each fiber section before the onset of reamination. Reamination was also evaluated after high-speed treadmill running with or without inhibition of reamination by hadacidin. Running resulted in large accumulations of IMP in FTW and FTR fibers (3.5 and 1.4 mumul/g, respectively); IMP in FTR fibers was higher with hadacidin treatment. Reamination after running was much greater in FTR than in FTW fibers and was associated with recovery of phosphocreatine. After running, the purine degradation products inosine and hypoxanthine were increased in FTW and FTR fibers in normal and hadacidin-treated animals. Plasma inosine, hypoxanthine, and urate increased after exercise; concentrations continued to increase if reamination was inhibited by hadacidin. These results demonstrate that when muscle IMP is increased, subsequent degradation and loss of purines occur. Rapid reamination should minimize the quantity of purine lost from muscle and limit the metabolic cost of replenishing purines by the de novo synthesis or salvage pathways.
Nonsteroidal anti-inflammatory drugs alter chloride and fluid transport in bovine retinal pigment epithelium
Authors: Bialek S. , Quong J. N. , Yu K. , Miller S. S.
Published: December 2017

Nonsteroidal anti-inflammatory drugs (NSAIDs) were added to the solutions bathing the apical membrane of bovine retinal pigment epithelium (RPE)-choroid explants. For example, niflumic acid (100 microM) depolarized the basolateral membrane voltage (VB) by approximately 12 mV, increased transepithelial potential by 4.5 mV, decreased intracellular Cl activity by 13 mM, decreased transepithelial resistance by 17 omega.cm2, and increased the ratio of apical to basolateral membrane resistance nearly threefold. All of these changes are consistent with an increase in basolateral membrane Cl conductance. In addition, niflumic acid caused intracellular Ca concentration to decrease by 16 nM and fluid transport rate to increase by 1.5 microliters.cm-2.h-1. Flufenamic acid, which is structurally very similar to niflumic acid, had the opposite effects on membrane voltage and resistance. Basal application of the Cl channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or current clamping VB to the reversal potential for Cl practically abolished the niflumic acid response. The niflumic acid results suggest that certain NSAIDs can directly alter Cl conductance in the bovine RPE, apparently independently of cyclooxygenase inhibition.
Filled pore approximation: a theoretical framework for solute-solvent coupling in narrow water channels
Authors: Welling D. J. , Welling P. A. , Welling L. W.
Published: December 2017

A phenomenological model is presented of water and solute transport that is applicable to water pores with radii less than approximately 2 A. This includes such examples as gramicidin A, the proximal tubule basolateral membrane, and the aquaporin 1 (CHIP28) water channel. The model differs from the conventional single-file model by allowing for a variation of unoccupied volume within the pores. It is shown that the accessible or free portion of the unoccupied volume can be related to the mechanical frictional coefficients and thereby to the filtration and diffusive permeabilities by the filled pore approximation. In general, the smallness of the unoccupied volume represents the compactness of the molecules within the pore and is indicative of the single-file character of the motion of water and solute moving together. When that volume is equal to a single water volume, the results are identical to the conventional single-file model. An important result is that, despite very low diffusive permeabilities, the reflection coefficient of a solute can remain at approximately 0.5 if its frictional interaction with the channel walls is comparable with its frictional interaction with neighboring water molecules. This is consistent with values previously reported for NaCl in cell membranes of proximal tubule. The model predicts a minimum effective pore radius for a water channel of 1.78 A and corresponds to a maximum filtration-to-diffusion permeability ratio that is proportional to the length of the effective pore or channel. This limiting condition corresponds to a water channel completely filled by water and may be applicable to the aquaporin 1 water channel.