bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of intestinal Cl- secretion by clotrimazole: direct effect on basolateral membrane K+ channels
Authors: Devor D. C. , Singh A. K. , Gerlach A. C. , Frizzell R. A. , Bridges R. J.
Published: December 2017

We evaluated the effects of clotrimazole and clofibrate on Ca(2+)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO) to activate the Ca(2+)-dependent K+ channel (KCa) in these cells to induce a sustained Cl- secretory current (Isc). Clotrimazole potently inhibited the KCa-dependent Isc, with an inhibition constant (Ki) of 0.27 +/- 0.02 microM. Clofibrate also inhibited the 1-EBIO-induced Isc albeit with lower affinity (Ki = 6.5 +/- 1.2 microM). Clotrimazole (10 microM) inhibited the Isc response to the Ca(2+)-mediated agonist, carbachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with Ki values of 5.2 +/- 1.0 and 6.7 +/- 1.1 microM, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and clofibrate on the basolateral K+ (IK) and apical Cl- (ICl) currents following stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced IK without affecting ICl. We determined the effects of clotrimazole and clofibrate on KCa using 86Rb+ uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced 86Rb+ uptake, with Ki values of 0.31 +/- 0.08 and 10.8 +/- 5.5 microM, respectively. Similarly, clotrimazole inhibited the Ca(2+)-induced 86Rb+ uptake with a Ki of 0.51 +/- 0.15 microM. Charybdotoxin inhibited both the 1-EBIO- and Ca(2+)-induced 86Rb+ uptakes with similar affinities (Ki values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggesting 1-EBIO and Ca2+ activate the same channel (KCa) in this assay. In excised, single-channel recordings both clotrimazole and clofibrate inhibited KCa, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal KCa, thereby inhibiting Cl- secretion. These results suggest that clotrimazole may be useful as an antidiarrheal.
Contribution of a swelling-activated chloride current to changes in the cardiac action potential
Authors: Vandenberg J. I. , Bett G. C. , Powell T.
Published: December 2017

The purpose of this investigation was to determine to what extent the swelling-activated Cl- current (ICl,swell) contributes to swelling-induced changes in the resting membrane potential and action potential duration (APD) in ventricular myocytes. Action potentials were recorded from guinea pig ventricular myocytes using conventional whole cell recording techniques. Cell swelling caused initial lengthening followed by a variable shortening of APD. In 59% of cells this secondary APD shortening had a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive component, consistent with a contribution from ICl,swell. Furthermore, DIDS partially antagonized the depolarization of the resting membrane potential that occurred during cell swelling. We have modeled the ICl,swell using the Oxsoft Heart computer program. Action potential changes predicted by the model agree well with the observed DIDS-sensitive component of the change in the action potential during cell swelling. We conclude that activation of ICl,swell contributes to shortening of APD and depolarization of the resting membrane potential during cell swelling in cardiac myocytes.
Differential effects of creatine kinase isoenzymes and substrates on regeneration in livers of transgenic mice
Authors: Askenasy N. , Koretsky A. P.
Published: December 2017

Creatine kinase (CK) has been implicated in affecting cell growth, and the CK substrates creatine (Cr) and cyclocreatine (CyCr) have been shown to have anti-tumor activity. The influence of Cr and CyCr on liver regeneration following major hepatectomy was evaluated in normal and transgenic mice expressing the human ubiquitous mitochondrial isoform of CK (CK-mit) or the brain isoform of CK (CK-B) or livers expressing both CK-mit and CK-B (CK-comb). Expression of CK isoenzymes had little effect on liver regeneration in the absence of dietary supplementation with Cr or CyCr as assayed by the increase in liver mass. Dietary supplementation with Cr and CyCr significantly reduced liver growth in normal mice. Liver regeneration was almost completely inhibited in mice expressing CK-mit in the presence of Cr. Livers expressing CK-mit regenerated better than normal livers in the presence of CyCr. In mice expressing CK-B, Cr and CyCr had opposite effects from those found in CK-mit mice. In the presence of CyCr, regeneration was inhibited in livers expressing CK-B, and, in the presence of Cr, CK-B-expressing livers regenerated better than normal livers. The amount of DNA synthesized 2 days after hepatectomy confirmed the results obtained from measurements of liver mass for all groups. Growth and DNA synthesis were completely abolished by Cr in CK-mit mice, whereas CyCr mainly affected growth 2 days after hepatectomy in CK-B-expressing mice. Coexpression of the CK isoforms in CK-comb mice ameliorated the effects detected with either isoform alone. Inhibition of growth by Cr and CyCr was not correlated to water accumulation. These results clearly demonstrate isoenzyme and substrate-specific effects of CK on cell growth.
CD14-dependent mechanism for endotoxin-mediated nitric oxide synthesis in murine macrophages
Authors: Schroeder R. A. , delaTorre A. , Kuo P. C.
Published: December 2017

Endotoxin-mediated macrophage synthesis of nitric oxide (NO) is associated with immune effector function, intercellular communication, leukocyte adhesion, vascular integrity, and neurotransmission. However, little is known of the cellular receptor and signal transduction pathway by which endotoxin induces NO production. With the use of a model of ANA-1 murine macrophages, we stimulated NO production by incubation with increasing concentrations of endotoxin and 5% fetal calf serum. In selected instances, the anti-CD14 antibody, ED9, was added. Endotoxin-mediated NO synthesis was dependent on CD14 function and the presence of an additional serum factor. Endotoxin treatment increased plasma membrane GTPase activity and 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) binding. Conversely, coincubation of cells with endotoxin and the heterotrimeric G protein inhibitors, suramin and guanosine 5'-O-(2-thiodiphosphate) trilithium salt, was associated with decreased NO synthesis, plasma membrane GTPase activity, and [35S]GTP gamma S binding. Blockade of CD14 or G protein function was associated with ablation of endotoxin-mediated inducible NO synthase (iNOS) protein expression, iNOS mRNA levels, and iNOS gene transcription, as determined by immunoblot, reverse transcriptase-polymerase chain reaction, and nuclear run-on analyses, respectively. These results indicate that endotoxin-mediated NO synthesis is a CD14-heterotrimeric G protein-dependent process.
Predicted changes in concentrations of free and bound ATP and ADP during intracellular Ca2+signaling
Authors: Kargacin M. E. , Kargacin G. J.
Published: December 2017

High Ca2+ concentrations can develop near Ca2+ sources during intracellular signaling and might lead to localized regulation of Ca2+-dependent processes. By shifting the amount of Ca2+ and other cations associated with ATP, local high Ca2+ concentrations might also alter the substrate available for membrane-associated and cytoplasmic enzymes. To study this, simultaneous equations were solved over a range of Ca2+ and Mg2+ concentrations to determine the general effects of Ca2+ on the concentrations of free and Ca2+- and Mg2+-bound forms of ATP. To obtain a more specific picture of the changes that might occur in smooth muscle cells, mathematical models of Ca2+ diffusion and regulation were used to predict the magnitude and time course of near-membrane Ca2+ transients and their effects on the free and bound forms of ATP near the membrane. The results of this work indicate that changes in free Ca2+ concentration over the range of 50 nM–100 μM would result in significant changes in free ATP concentration, MgATP concentration, and the CaATP-to-MgATP concentration ratio.
Role of prostaglandin H synthase-2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth
Authors: Martinez Javier , Sanchez Teresa , Moreno Juan J.
Published: December 2017

The specific role(s) of arachidonic acid (AA) and its metabolites in the signaling pathways that regulated fibroblast growth was studied. A Western blot analysis demonstrated that prostaglandin H synthase-2 (PGHS-2) was expressed by 3T6 fibroblast cultures in RPMI 1640 supplemented with fetal calf serum (10%). Dexamethasone, which inhibits AA release and PGHS-2 expression, significantly reduced cell proliferation. Ketoprofen, a dual cyclooxygenase inhibitor, and CGP-28238, a specific PGHS-2 inhibitor, reduced fibroblast proliferation in a dose-dependent manner. These drugs also reduced [3H]thymidine incorporation into the DNA of fibroblasts. These effects were correlated with a decrease in prostaglandin (PG) E2 levels in the cell medium. However, piroxicam at doses that selectively inhibit PGHS-1 did not have a significant effect on fibroblast proliferation. Finally, we showed that the antiproliferative effect of dexamethasone and PGHS-2 inhibitors was significantly antagonized when PGE2 was added to the culture medium. Our results suggest that PGHS-2 and prostaglandins such as PGE2 might play an important role in the regulation of 3T6 fibroblast growth stimulated by growth factors of serum.
Glycolysis inhibition by palmitate in renal cells cultured in a two-chamber system
Authors: Bolon Claire , Gauthier Catherine , Simonnet Hélène
Published: December 2017

A major shortcoming of renal proximal tubular cells (RPTC) in culture is the gradual modification of their energy metabolism from the oxidative type to the glycolytic type. To test the possible reduction of glycolysis by naturally occurring long-chain fatty acids, RPTC were cultured in a two-chamber system, with albumin-bound palmitate (0.4 mM) added to the basolateral chamber after confluency. Twenty-four hours of contact with palmitate decreased glycolysis by 38% provided that carnitine was present; lactate production was decreased by 38%, and the decrease in glycolysis resulted from a similar decrease of basolateral and apical net uptake of glucose. In contrast to the previously described effect of the nonphysiological oxidative substrate heptanoate, palmitate promoted a long-term decrease in lactate production and sustained excellent cellular growth. After 4 days of contact, decreased glycolysis was maintained even in the absence of carnitine and resulted from a decrease of basolateral uptake only, suggestive of long-term regulation different from the earlier effects. Thus, although cultured RPTC lost their oxidative phenotype, they exhibited a type of regulation (Randle effect) that is found in the oxidative-type but not in the glycolytic-type tissues, therefore unmasking a regulative capacity barely detectable in fresh RPTC. Low Po 2 (50 mmHg in the apical chamber) could be a major cause of elevated glycolysis and could hinder the effects of palmitate.
Reappearance of the minor α-sarcomeric actins in postnatal muscle
Authors: Collins Teresa , Joya Josephine E. , Arkell Ruth M. , Ferguson Vicki , Hardeman Edna C.
Published: December 2017

The postnatal expression profiles of α-sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. α-Cardiac actin (α-CA) transcripts transiently increase between 12 and 21 days after birth in the quadriceps muscle, reaching ∼90% that found in the adult mouse heart. Although α-CA is the α-sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that this postnatal period is not reflective of an immature phenotype. α-Skeletal actin (α-SA) transcripts accumulate to ∼32% of the total α-sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of α-CA and α-SA in postnatal skeletal and heart muscles, respectively, and 2) the contractile protein gene expression profile characteristic of adult skeletal muscle is not achieved until after 42 days postnatal in the mouse. We propose there is a previously uncharacterized period of postnatal striated muscle maturation marked by the reappearance of the minor α-sarcomeric actins.
Downregulation of caveolin by chronic β-adrenergic receptor stimulation in mice
Authors: Oka Naoki , Asai Kuniya , Kudej Raymond K. , Edwards John G. , Toya Yoshiyuki , Schwencke Carsten , Vatner Dorothy E. , Vatner Stephen F. , Ishikawa Yoshihiro
Published: December 2017

Caveolae, flask-shaped invaginations of cell membranes, are believed to play pivotal roles in transmembrane transportation of molecules and cellular signaling. Caveolin, a structural component of caveolae, interacts directly with G proteins and regulates their function. We investigated the effect of chronic β-adrenergic receptor stimulation on the expression of caveolin subtypes in mouse hearts by immunoblotting and Northern blotting. Caveolin-1 and -3 were abundantly expressed in the heart and skeletal muscles, but not in the brain. Continuous (−)-isoproterenol, but not (+)-isoproterenol, infusion via osmotic minipump (30 μg ⋅ g−1 ⋅ day−1) for 13 days significantly downregulated both caveolin subtypes in the heart. The expression of caveolin-1 was reduced by 48 ± 6.1% and that of caveolin-3 by 28 ± 4.0% ( P < 0.01, n = 8 for each). The subcellular distribution of caveolin subtypes in ventricular myocardium was not altered as determined by sucrose gradient fractionation. In contrast, the expression of both caveolin subtypes in skeletal muscles was not significantly changed. Our data suggest that the expression of caveolin subtypes is regulated by β-adrenergic receptor stimulation in the heart.
Effect of IBMX and alkaline phosphatase inhibitors on Cl− secretion in G551D cystic fibrosis mutant mice
Authors: Smith Stephen N. , Delaney Stephen J. , Dorin Julia R. , Farley Raymond , Geddes Duncan M. , Porteous David J. , Wainwright Brandon J. , Alton Eric W. F. W.
Published: December 2017

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl− channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl− secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl− secretion in cftrG551D mutant mice. No induction of Cl−secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means ± SE: jejunum, 1.8 ± 0.9 μA/cm2, n = 7; cecum, 3.7 ± 0.8 μA/cm2, n = 7; rectum (in vivo), 1.9 ± 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice ( cftrUNC ). In the trachea, IBMX produced a transient Cl− secretory response (37.3 ± 14.7 μA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 ± 4.7 μA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl−secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl− secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.
Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation
Authors: Furukawa Tetsushi , Ogura Takehiko , Katayama Yoshifumi , Hiraoka Masayasu
Published: December 2017

In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of ×40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl− ≥ Br− ≫ I− ≥ F−. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at −160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2ΔNT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2ΔNT-injected oocytes (−5.9 ± 0.4 μA), followed by ClC-2-injected oocytes (−4.3 ± 0.6 μA), and smallest in water-injected oocytes (−0.2 ± 0.2 μA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2ΔNT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to a hyposmolar environment.
Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts
Authors: Padua Rodolfo A. , Baron Kyle T. , Thyagarajan Bhaskar , Campbell Colin , Thayer Stanley A.
Published: December 2017

Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2+-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 ± 0.2 μM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mtincrease, although it did not affect the [Ca2+]ctransient. Basal [Ca2+]cand [Ca2+]mtin control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mttransients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.
Circadian regulation of CREB transcription factor in mouse esophagus
Authors: Scheving Lawrence A. , Gardner Wendi
Published: December 2017

Very little is known about the circadian regulation of cell entry into the S and M phases of the cell cycle. Yet, in the mouse esophagus, a seven- to ninefold increase in DNA synthesis coincides with nocturnal feeding. The phosphorylation of the cAMP response element binding protein (CREB), a transcriptional factor, may regulate hypothalamic circadian rhythms in the brain. Here, we investigate the circadian regulation of CREB and Ser-133-phospho-CREB (PCREB) in the mouse esophagus by immunocytochemical and biochemical methods. We found that, during the dark phase, coincident with the onset of feeding and increased DNA synthesis, esophageal CREB and PCREB expression decreased. Although CREB-like immunoreactivity (CREB-lir) was expressed in many different cell types, it was concentrated in the mucosa, particularly in the replicating basal cell layer. The injection of epidermal growth factor, at a dosage known to maximally stimulate esophageal DNA synthesis in a 4- to 8-h period, rapidly decreased PCREB levels within 10 min of injection. We speculate that PCREB-lir may be involved in the circadian regulation of cell cycle events in the intact mouse esophagus.
A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin
Authors: Smith Aiping F. , Bigsby Robert M. , Word R. Ann , Herring B. Paul
Published: December 2017

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from −163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (−2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between −1447 and −1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between −1447 and −1474.
Human Na+-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts
Authors: Porcellati Francesca , Hlaing Tommy , Togawa Masaki , Stevens Martin J. , Larkin Dennis D. , Hosaka Yoshiyuki , Glover Thomas W. , Henry Douglas N. , Greene Douglas A. , Killen Paul D.
Published: December 2017

Na+- myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na+- myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na+-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and ∼1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.
Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice
Authors: Tullson Peter C. , Rush James W. E. , Wieringa Bé , Terjung R. L.
Published: December 2017

Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK−/−) or also deficient in mitochondrial creatine kinase (CK−/−) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK−/− and CK−/− mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 ( AMPD1) gene expression was unchanged. Kinetic analysis of AMP deaminase from mixed muscle revealed negative cooperativity that was significantly greater in creatine kinase deficiencies. Treatment of AMP deaminase with acid phosphatase abolished negative cooperative behavior, indicating that a phosphorylation-dephosphorylation cycle may be important in the regulation of AMP deaminase.
Release of dopamine and norepinephrine by hypoxia from PC-12 cells
Authors: Kumar Ganesh K. , Overholt Jeffrey L. , Bright Gary R. , Hui Kwong Y. , Lu Hongwen , Gratzl Miklos , Prabhakar Nanduri R.
Published: December 2017

We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (Po 2 of medium ∼40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of Po 2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was ∼11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 μM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in Po 2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.
Synergism between toxin-γ from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells
Authors: Conceiçao Isaltino M. , Lebrun Ivo , Cano-Abad María , Gandía Luis , Hernández-Guijo Jesús M. , López Manuela G. , Villarroya Mercedes , Jurkiewicz Aron , García Antonio G.
Published: December 2017

Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tγ was ∼200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tγ (1.2 vs. 12 μg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+concentration ([Ca2+]o), when 30 μM veratridine plus 0.45 μM Tγ were used. Tγ (0.45 μM) doubled the basal uptake of45Ca2+, whereas veratridine (100 μM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tγ and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tγ elevated basal [Ca2+]ilevels, causing only small oscillations. When added together, Tγ and veratridine elevated the basal levels of [Ca2+]iwithout causing large oscillations. Tγ shifted the current-voltage ( I-V) curve for Na+ channel current to the left. The combination of Tγ with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.
Ion channels in microglia (brain macrophages)
Authors: Eder Claudia
Published: December 2017

Microglia are immunocompetent cells in the brain that have many similarities with macrophages of peripheral tissues. In normal adult brain, microglial cells are in a resting state, but they become activated during inflammation of the central nervous system, after neuronal injury, and in several neurological diseases. Patch-clamp studies of microglial cells in cell culture and in tissue slices demonstrate that microglia express a wide variety of ion channels. Six different types of K+ channels have been identified in microglia, namely, inward rectifier, delayed rectifier, HERG-like, G protein-activated, as well as voltage-dependent and voltage-independent Ca2+-activated K+ channels. Moreover, microglia express H+ channels, Na+ channels, voltage-gated Ca2+ channels, Ca2+-release activated Ca2+ channels, and voltage-dependent and voltage-independent Cl− channels. With respect to their kinetic and pharmacological properties, most microglial ion channels closely resemble ion channels characterized in other macrophage preparations. Expression patterns of ion channels in microglia depend on the functional state of the cells. Microglial ion channels can be modulated by exposure to lipopolysaccharide or various cytokines, by activation of protein kinase C or G proteins, by factors released from astrocytes, by changes in the concentration of internal free Ca2+, and by variations of the internal or external pH. There is evidence suggesting that ion channels in microglia are involved in maintaining the membrane potential and are also involved in proliferation, ramification, and the respiratory burst. Further possible functional roles of microglial ion channels are discussed.
Effects of BAPTA on force and Ca2+ transient during isometric contraction of frog muscle fibers
Authors: Sun Y.-B. , Caputo C. , Edman K. A. P.
Published: December 2017

The effects of 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA) on force and intracellular Ca2+ transient were studied during isometric twitches and tetanuses in single frog muscle fibers. BAPTA was added to the bathing solution in its permeant AM form (50 and 100 μM). There was no clear correlation between the changes in force and the changes in Ca2+ transient. Thus during twitch stimulation BAPTA did not suppress the Ca2+ transient until the force had been reduced to <50% of its control value. At the same time, the peak myoplasmic free Ca2+concentration reached during tetanic stimulation was markedly increased, whereas the force was slightly reduced by BAPTA. The effects of BAPTA were not duplicated by using another Ca2+ chelator, EGTA, indicating that BAPTA may act differently as a Ca2+ chelator. Stiffness measurements suggest that the decrease in mechanical performance in the presence of BAPTA is attributable to a reduced number of active cross bridges. The results could mean that BAPTA, under the conditions used, inhibits the binding of Ca2+ to troponin C resulting in a reduced state of activation of the contractile system.
Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells
Authors: Singh A. K. , Taskén K. , Walker W. , Frizzell R. A. , Watkins S. C. , Bridges R. J. , Bradbury N. A.
Published: December 2017

Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIα and RIIα. In addition, immunogold electron microscopy showed that RIIα labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIα was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Cα was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (∼100 μA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.
Exocytosis is not involved in activation of Cl− secretion via CFTR in Calu-3 airway epithelial cells
Authors: Loffing Johannes , Moyer Bryan D. , McCoy David , Stanton Bruce A.
Published: December 2017

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel, which mediates transepithelial Cl− transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl− secretion in part by increasing the number of CFTR Cl− channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl− secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl− secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl− channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl− secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl− secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.
Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7channel
Authors: Wiley J. S. , Gargett C. E. , Zhang W. , Snook M. B. , Jamieson G. P.
Published: December 2017

Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3′- O-(4-benzoyl)benzoyl-adenosine 5′-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5′-triphosphate, or adenosine 5′- O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 μM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 μM) at 37°C, and the delay was longer at 24°C. ATP addition 2–10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10–20 μM). P2Z/P2X7 inhibitors, KN-62 and 5-( N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.
Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis
Authors: Dastych Jaroslaw , Taub Dennis , Hardison Mary C. , Metcalfe Dean D.
Published: December 2017

W/Wv mice are deficient in tissue mast cells, and mast cells cultured from these mice do not proliferate in response to the c-kit ligand, stem cell factor (SCF). In this paper, we report that mouse bone marrow cultured mast cells derived from W/Wv mice do adhere to fibronectin in the presence of SCF and exhibit chemotaxis to SCF, and we explore this model for the understanding of c-kit-mediated signaling pathways. Both in vitro and in vivo (in intact cells) phosphorylation experiments demonstrated a low residual level of W/Wv c-kit protein phosphorylation. SCF-induced responses in W/Wv mast cells were abolished by the tyrosine kinase inhibitor herbimycin A and by the phospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin but were not affected by protein kinase C inhibitors. These observations are consistent with the conclusions that Wv c-kit initiates a signaling process that is PI 3-kinase dependent and that mutated Wv c-kit retains the ability to initiate mast cell adhesion and migration.
Na+ and K+ regulate the phosphorylation state of nucleoside diphosphate kinase in human airway epithelium
Authors: Marshall L. J. , Muimo R. , Riemen C. E. , Mehta A.
Published: December 2017

We describe how cations, in the presence of ATP, regulate the phosphorylated form of 19- and 21-kDa nucleoside diphosphate kinase (NDPK; EC 2.7.4.6 ), a kinase controlling K+ channels, G proteins, cell secretion, cellular energy production, and UTP synthesis. In apically enriched human nasal epithelial membranes, 10 mM Na+ inhibits phosphorylation of NDPK relative to other cations. Dose response showed that, whereas K+ induces a fourfold greater phosphate incorporation (EC50 10 mM), Na+ is inhibitory (EC50 10 mM) compared with respective buffer controls. Cation discrimination is nucleotide selective (not seen with [γ-32P]GTP) and NDPK specific (not seen with p37h, a previously characterized Cl−-sensitive phosphoprotein). Na+ does not exert an inhibitory effect on NDPK phosphorylation directly but is likely to act via an okadaic acid-insensitive phosphatase. We speculate that the ability of NDPK to discriminate between physiologically relevant cation concentrations provides a novel example of cross talk within the apical membrane.
Dynamics of calcium regulation of chloride currents inXenopus oocytes
Authors: Kuruma Akinori , Hartzell H. Criss
Published: December 2017

Ca-activated Cl currents are widely expressed in many cell types and play diverse and important physiological roles. The Xenopus oocyte is a good model system for studying the regulation of these currents. We previously showed that inositol 1,4,5-trisphosphate (IP3) injection into Xenopus oocytes rapidly elicits a noninactivating outward Cl current ( I Cl1-S) followed several minutes later by the development of slow inward ( I Cl2) and transient outward ( I Cl1-T) Cl currents. In this paper, we investigate whether these three currents are mediated by the same or different Cl channels. Outward Cl currents were more sensitive to Ca than inward Cl currents, as shown by injection of different amounts of Ca or by Ca influx through a heterologously expressed ligand-gated Ca channel, the ionotropic glutamate receptor iGluR3. These data could be explained by two channels with different Ca affinities or one channel with a higher Ca affinity at depolarized potentials. To distinguish between these possibilities, we determined the anion selectivity of the three currents. The anion selectivity sequences for the three currents were the same (I > Br > Cl), but I Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, after consideration of possible errors, the hypothesis that a single type of Cl channel underlies the complex waveforms of the three different macroscopic Ca-activated Cl currents in Xenopus oocytes remains a viable alternative.
Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells
Authors: Gillen Christopher M. , Forbush Bliss
Published: December 2017

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.
Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells
Authors: Collard Charles D. , Bukusoglu Cuneyt , Agah Azin , Colgan Sean P. , Reenstra Wende R. , Morgan B. Paul , Stahl Gregory L.
Published: December 2017

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 ± 0.6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-α also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.
Polyamine depletion alters the relationship of F-actin, G-actin, and thymosin β4 in migrating IEC-6 cells
Authors: McCormack Shirley A. , Ray Ramesh M. , Blanner Patrick M. , Johnson Leonard R.
Published: December 2017

The cause of reduced migration ability in polyamine-deficient cells is not known, but their actin cytoskeleton is clearly abnormal. We depleted polyamines with α-difluoromethylornithine (DFMO) in migrating cells with or without stimulation by epidermal growth factor (EGF) and investigated filamentous (F-) actin, monomeric (G-) actin, and thymosin β4 (Tβ4), using immunofluorescent confocal microscopy, DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cell interior, increased it in the cell cortex, redistributed G-actin, and increased nuclear staining of Tβ4. However, DFMO did not affect the amount of Tβ4 mRNA. EGF caused a rapid increase in the staining of F-actin in control cells, but DFMO prevented this response to EGF. Despite the visible changes shown by immunocytochemistry, statistically significant changes in the amount of either actin isoform or of total actin did not occur. We propose that DFMO reduces migration by interfering with the sequestration of G-actin by Tβ4 and the association of F-actin with activated EGF receptors.
Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR
Authors: Rolfe David F. S. , Newman John M. B. , Buckingham Julie A. , Clark Michael G. , Brand Martin D.
Published: December 2017

Proton pumping across the mitochondrial inner membrane and proton leak back through the natural proton conductance pathway make up a futile cycle that dissipates redox energy. We measured respiration and average mitochondrial membrane potential in perfused rat hindquarter with maximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similar measurements in rat hepatocytes given substrates to cause a high rate of gluconeogenesis and ureagenesis showed that the proton cycle accounted for 22% of the respiration rate of these cells. Combining these in vitro values with literature values for the contribution of skeletal muscle and liver to standard metabolic rate (SMR), we calculate that the proton cycle in working muscle and liver may account for 15% of SMR in vivo. Although this value is less than the 20% of SMR we calculated previously using data from resting skeletal muscle and hepatocytes, it is still large, and we conclude that the futile proton cycle is a major contributor to SMR.