bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological activation changes stiffness of cultured human airway smooth muscle cells
Authors: Hubmayr R. D. , Shore S. A. , Fredberg J. J. , Planus E. , Panettieri R. A. , Moller W. , Heyder J. , Wang N.
Published: December 2017

Using magnetic twisting cytometry (MTC), we measured the cytoskeletal stiffness of adherent human airway smooth muscle (HASM) cells. We hypothesized that modulation of actin-myosin interactions by application of contractile agonists would induce changes in cytoskeletal stiffness. In cells plated on high-density collagen, bradykinin (10(-6) M) and histamine (10(-4) M) increased stiffness by 85 +/- 15 and 68 +/- 16%, respectively. Increases in cell stiffness were also consistently observed after acetylcholine, substance P, and KCl. The bronchodilator agonists isoproterenol, prostaglandin E2, forskolin, dibutryl adenosine 3', 5'-cyclic monophosphate, and 8-bromoguanosine 3', 5'-cyclic monophosphate each caused a dose-dependent decrease in cell stiffness in unstimulated as well as bradykinin-treated cells. HASM cells plated on high-density collagen were stiffer than cells plated on low-density collagen (126 +/- 16 vs. 43 +/- 3 dyn/cm2) and developed more pronounced increases in stiffness in response to bradykinin as well as more pronounced decreases in stiffness in response to isoproterenol. These results are consistent with the hypothesis that modulation of actin-myosin interactions by application of contractile agonists causes changes in cytoskeletal stiffness of HASM cells. MTC may be a valuable tool for evaluating the mechanisms of pharmacomechanical coupling in airway smooth muscle cells in culture.
Actin polymerization and depolymerization during apoptosis in HL-60 cells
Authors: Levee M. G. , Dabrowska M. I. , Lelli J. L. , Hinshaw D. B.
Published: December 2017

Little is known about the biochemical “machinery” responsible for the morphological features of apoptosis, although the cytoskeleton is presumed to be involved. Using flow cytometry, polyacrylamide gel electrophoresis, and fluorescence microscopy, we show that apoptosis induced by ultraviolet (UV) irradiation or 80 micrograms/ml etoposide correlates with early transient polymerization and later depolymerization of filamentous (F)-actin and dramatic changes in visible microfilament organization. Depolymerization of F-actin began before the formation of apoptotic bodies and was ultimately composed of decreases in both the detergent-insoluble (40%) and detergent-soluble (50%) pools of F-actin. Dihydrocytochalasin B (H2CB), which blocked apoptotic body formation, depolymerized F-actin in the detergent-insoluble pool only. Visually, H2CB treatment disrupted microfilament organization, resulting in short, brightly stained microfilaments dispersed throughout the cytoplasm. In contrast, apoptotic cells contained a network of fine microfilaments with bright staining concentrated at the site of apoptotic body formation. Together, these results suggest that reorganization of the microfilament network is necessary for the formation of apoptotic bodies and that depolymerization of F-actin may also be a necessary component of the process of apoptosis.
Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion
Authors: Zoukhri D. , Hodges R. R. , Sergheraert C. , Toker A. , Dartt D. A.
Published: December 2017

In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.
Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform
Authors: Chambrey R. , Achard J. M. , Warnock D. G.
Published: December 2017

Molecular cloning and expression have previously defined three members of the Na+/H+ exchanger (NHE) gene family NHE1 and NHE2 are sensitive to inhibition by amiloride and its 5'-amino alkyl-substituted analogues, whereas NHE3 is quite resistant to amiloride inhibition. Each of these exchangers has narrowly defined cation specificities for Na+ and Li+. Expression studies with NHE4 have not been as successful, with only a description of modest expression of activity (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994). We now report that NHE4 activity in stably transfected fibroblasts is activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), permitting functional characterization of this NHE isoform. The activating effect of DIDS was unique among the disulfonic stilbenes, and competition studies suggested a cross-linking mechanism. NHE4 is extremely resistant to amiloride and ethylisopropylamiloride inhibition and, unlike other NHE isoforms, affects K+/H+ exchange as well as Na+/H+ and Li+/H+ exchange. These findings demonstrate that NHE4 is a functionally distinct member of the NHE gene family and suggest a unique physiological role for this cation/H+ exchanger.
Differential expression and activation of MAP kinases in dedifferentiated MDCK-focus cells
Authors: Schramek H. , Schumacher M. , Wilflingseder D. , Oberleithner H. , Pfaller W.
Published: December 2017

Mitogen-activated protein kinases (MAPK) play a key role in the regulation of cellular processes such as cell growth, cell differentiation, and apoptosis. However, the specific function of single isoforms of the MAPK family in renal epithelial cell differentiation and/or proliferation has not been investigated so far. We now report stable reduction of extracellular signal-regulated kinase 1 (ERK1) protein expression and lack of serum-induced ERK1 activation in alkali-dedifferentiated Madin-Darby canine kidney-C7 focus (MDCK-C7F) cells compared with their parental epithelial MDCK-C7 cells. The changes in ERK1 protein expression and activation were accompanied by a small rise in c-jun NH2-terminal kinase 1 (JNK1) protein expression but slightly decreased basal and anisomycin-stimulated JNK1 activity. In contrast, ERK2 phosphorylation, as assessed by using an antibody which detects phosphorylated tyrosine 204 of both ERK1 and ERK2, as well as enzymatic ERK2 activity, was substantially increased in untreated and fetal calf serum-treated MDCK-C7F cells, although ERK2 protein expression remained unchanged. Differential expression and activation of ERK1, ERK2, and JNK1 were accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation. Together, our results demonstrate an association between changes in the activation of certain MAPK and alkali-induced stable MDCK-C7 cell dedifferentiation. Moreover, these data provide evidence for distinct signaling functions of ERK1 and ERK2 in these cells.
Mechanisms of alpha2-adrenoceptor-mediated inhibition in rabbit carotid body
Authors: Almaraz L. , Perez-Garcia M. T. , Gomez-Nino A. , Gonzalez C.
Published: December 2017

We have used the in vitro preparation of the intact carotid body (CB) and isolated chemoreceptor cells to elucidate the distribution and function of alpha2-adrenoreceptors. The significance of the study lies in the fact that norepinephrine (NE), being the neurotransmitter of the sympathetic innervation to the CB, is also abundant in chemoreceptor cells. In intact CB whose catecholamine (CA) deposits had been labeled by prior incubation with the CA precursor [3H]tyrosine, the alpha2-antagonist yohimbine (10 microM) potentiated the low-PO2 (33 and 60 mmHg)-induced release of [3H]CA by 100 and 53%, respectively. Yohimbine (10 microM) and SKF-86466 (50 microM; another alpha2-antagonist) reversed the inhibition of the release of [3H]CA produced by the alpha2-receptor agonists clonidine and UK-14304 (10 microM). The increase in adenosine 3',5'-cyclic monophosphate produced by low PO2 was further augmented by yohimbine and nearly halved by UK-14304 and clonidine. In isolated chemoreceptor cells, UK-14304 and NE inhibited voltage-dependent Ca2+ currents by 28 and 32%, respectively. These results indicate that alpha2-receptors are present in chemoreceptor cells, where they reduce the release of [3H]CA. Inhibition of adenylate cyclase(s) and Ca2+ channels may be involved in this effect. Using intact CB from normal and chronically sympathectomized animals, we demonstrated a specific accumulation of [3H]NE in intraglomic sympathetic endings. Hypoxia (PO2 approximately 33 mmHg) did not elicit release of [3H]NE from the sympathetic endings, but high extracellular K+ (K+(e)) induced a release of [3H]NE that was inhibited by alpha2-agonists and augmented by alpha2-antagonists. These findings demonstrate that alpha2-receptors are also present in the sympathetic endings of the CB, where they modulate the release of NE. As a whole, this work provides a more detailed understanding of the role of the sympathetic innervation in the control of the CB chemoreceptor function, including the cellular mechanisms of the action of NE.
Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis
Authors: Fabisiak J. P. , Kagan V. E. , Ritov V. B. , Johnson D. E. , Lazo J. S.
Published: December 2017

Oxidative stress is a potential component of the final common pathway leading to apoptosis following many diverse stimuli. Here, we document that the oxidant paraquat caused apoptosis in mouse 32D cells. We examined early paraquat-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid (cis-PA) into phospholipids and high-performance liquid chromatography separation of specific phospholipid classes. Paraquat induced peroxidation of cis-PA primarily in phosphatidylserine (PS) and to a lesser extent in phosphatidylinositol (PI) within 2 h. The selective oxidation of PS occurred before signs of cytotoxicity and preceded the externalization of PS as assessed by annexin V binding. Overexpression of Bcl-2 afforded significant protection against paraquat-induced apoptosis, early PS and PI oxidation, and PS externalization but not the ultimate formation of high-molecular-weight DNA fragments. Therefore, both selective phospholipid peroxidation and DNA damage occurred after paraquat exposure, but only the former was specifically associated with apoptosis. We suggest Bcl-2 may inhibit oxidant-induced apoptosis by preventing the peroxidation of specific membrane phospholipids.
cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia
Authors: Schwiebert E. M. , Potter E. D. , Hwang T. H. , Woo J. S. , Ding C. , Qiu W. , Guggino W. B. , Levine M. A. , Guggino S. E.
Published: December 2017

To test the hypothesis that guanosine 3',5'-cyclic monophosphate (cGMP) regulates ion transport in airway epithelial cells, we measured short-circuit current (I(sc)) and (22)Na+ fluxes in primary cultured rat tracheal epithelial cells. In Cl- -containing Ringer solution, I(sc) was increased by approximately 17 microA/cm2 after application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), whereas, in Cl- -free solutions, the Na+ -mediated component was approximately 5 microA/cm2, suggesting a cGMP stimulation of Cl-secretory current and a smaller Na+ absorptive current. Inward and net mucosal-to-serosal (22)Na+ flux was doubled in the presence of 2 mM 8-BrcGMP. To determine whether nucleotide-gated channels play a role in this transepithelial Na+ absorption, blockers of nucleotide-gated cation channels were used to inhibit I(sc). The cGMP-stimulated Na+-mediated I(sc) was blocked by as little as 500 nM dichlorobenzamil or 50 microM L-cis-diltiazem, which are known blockers for cyclic nucleotide-gated cation channels. These agents also blocked the basal (non-cGMP-stimulated) current when measured in the presence of 10 microM amiloride, which blocks current through 5-pS amiloride-sensitive Na+ channels. To document whether the distribution of nucleotide-gated nonselective cation channels was consistent with a role in airway epithelial transport, in situ hybridization was performed. In situ hybridization of mRNA encoding for nucleotide-gated cation channels was found in epithelial cell layers of rat trachea, bronchi, bronchioles, and alveolar cells but not in smooth muscle layers or tracheal cartilage. Reverse transcriptase-polymerase chain reaction, restriction enzyme analysis, and sequencing of the cDNA transcribed from mRNA of whole lung and tracheal epithelial cells indicate that a channel highly homologous to the retinal nucleotide-gated nonselective cation channel (CNG1) is present. Thus these data, along with evidence supporting the existence of signal transduction pathways elevating intracellular levels of cGMP, indicate that cGMP regulates transepithelial ion transport in lung epithelial tissues.
Expression of functional mitochondrial creatine kinase in liver of transgenic mice
Authors: Miller K. , Sharer K. , Suhan J. , Koretsky A. P.
Published: December 2017

The mitochondrial isoform of creatine kinase (MiCK) is localized to the mitochondrial intermembrane space, and its precise role in vivo is still actively being investigated. Here, we report a transgenic mouse model in which MiCK is expressed in liver, a tissue that does not normally express significant levels of CK. Expression of the genomic clone for human, ubiquitous MiCK was controlled by the promoter/enhancer region of the transthyretin gene. Three of seven founder mice were chosen to establish lines and had MiCK activity values ranging from 13 to 269 micromol x min(-1) x g wet wt(-1). Differential centrifugation and histochemical staining demonstrated that >90% of the CK activity is localized to the mitochondrial intermembrane space. An unusual mitochondrial morphology characterized by an angular nature to the membranes was detected using electron microscopy in the transgenic line expressing the highest levels of MiCK. Increasing hepatic total creatine levels led to a return to normal mitochondrial morphology. 31P-nuclear magnetic resonance spectroscopy demonstrated that the expressed MiCK is capable of producing and utilizing phosphocreatine. These mice will be useful for investigating gain of function effects of MiCK in cellular energetics.
Mitochondrial biogenesis during cellular differentiation
Authors: Moyes C. D. , Mathieu-Costello O. A. , Tsuchiya N. , Filburn C. , Hansford R. G.
Published: December 2017

Mitochondrial biogenesis was studied during differentiation of two immortalized cell lines (C2C12, 3T3) with enzyme measurements, Northern blots, and quantitative ultrastructure. Citrate synthase, isocitrate dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase (nuclear encoded, mitochondrial matrix location) showed linear, four- to sixfold increases in enzymatic activity in C2C12 cells but increased exponentially in 3T3 cells. Cytochrome oxidase and NADH dehydrogenase (nuclear and mitochondrial encoded, cristae location) increased to a lesser extent and with a pattern dissimilar to the first group. Northern blots and activity of succinate dehydrogenase (cristae location but entirely nuclear encoded) suggested the groupings were based on location of the genes rather than the mature enzyme. However, quantitative electron microscopy and comparisons with adult tissue suggested that mitochondrial ultrastructure can influence the change in cristae enzymes. Cristae surface area per unit mitochondrial volume and per unit cell volume increased much less than did cristae enzymes. Available space on the inner membrane may become limiting and account for some aspects of the pattern of change in electron transport enzymes during differentiation.
Acute metabolic acidosis inhibits the induction of osteoblastic egr-1 and type 1 collagen
Authors: Frick K. K. , Jiang L. , Bushinsky D. A.
Published: December 2017

Metabolic acidosis induces net calcium efflux from bone through a decrease in osteoblastic formation and an increase in osteoclastic resorption. We tested the hypothesis that changes in external pH would alter the expression of genes critical to the function of mouse calvarial bone cells, predominantly osteoblasts. Cells were cultured in physiologically neutral pH medium until confluent and then stimulated with fresh medium at either neutral or acidic pH. Among a group of immediate early response genes, including egr-1, junB, c-jun, junD, and c-fos, only egr-1 stimulation was modulated by changes in medium pH. At pH 7.4, RNA for egr-1 was stimulated approximately 10- to 30-fold, 40 min after medium change. A progressive decrease in pH to 6.8 led to a parallel reduction in egr-1 stimulation, and an increase in pH to 7.6 led to an increase in egr-1 stimulation. The protein synthesis inhibitor cycloheximide led to a superinduction of egr-1 with preservation of the pH dependency of expression. Osteoblasts synthesize collagen, which is subsequently mineralized. RNA for type 1 collagen was stimulated approximately three- to fivefold, 40 min after medium change. Again the stimulation was inhibited by acidosis and increased by alkalosis. Cycloheximide abolished the pH dependency of expression. These results suggest that small changes in external pH have a significant effect on the expression of certain genes important for osteoblastic function.
NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries
Authors: DiSanto M. E. , Cox R. H. , Wang Z. , Chacko S.
Published: December 2017

We demonstrate, using reverse transcriptase-polymerase chain reaction, that, whereas abdominal aorta from rabbit consists almost entirely of myosin heavy chain (MHC) mRNA with no insert at the 5'-terminal coding region, the distributing arteries (femoral and saphenous) begin to show MHC mRNA with the 21-nucleotide insert that encodes seven amino acids in the ATP-binding region located in the myosin head. The femoral/iliac artery contains > 50% inserted mRNA, whereas the more distal saphenous artery contains > 80% inserted mRNA. This insert is also present in the smooth muscle from rat tail artery but is absent in the smooth muscle from rat aorta. The actin-activated ATPase activity of myosin from the rabbit femoral/saphenous artery is 1.7-fold higher than that of the myosin from the aorta. A concomitant increase (about twofold) in the maximum shortening velocity of the saphenous artery, compared with that of the aorta, indicates that the preponderance of the inserted myosin is associated with both an increase in the actin-activated ATPase activity and a larger maximum velocity of shortening. Furthermore, analysis of the 17-kDa essential light chain from the aorta reveals near equal quantities of the 17-kDa light chain isoforms a and b, whereas the myosin from the femoral/ saphenous artery contains predominantly the 17-kDa light chain a isoform. Together, these data indicate that the smooth muscle cells from the small distributing arteries are similar to those of visceral smooth muscle with respect to the expression of myosin isoforms, actin-activated myosin ATPase activity and contractility.
In vivo brain phosphocreatine and ATP regulation in mice fed a creatine analog
Authors: Holtzman D. , Meyers R. , O'Gorman E. , Khait I. , Wallimann T. , Allred E. , Jensen F.
Published: December 2017

Mitochondrial and cytosolic creatine kinase (CK) isozymes are active in cells with high and variable ATP metabolic rates. beta-Guanidinopropionic acid (GPA), a competitive inhibitor of creatine transport, was used to study the hypothesis that the creatine-CK-phosphocreatine (PCr) system is important in regulating brain ATP metabolism. The CK-catalyzed reaction rate and reactant concentrations were measured in vivo with 31P nuclear magnetic resonance spectroscopy during energy deficit (hypoxia) or high-energy turnover (seizures) states in urethane-anesthetized mice fed GPA, creatine, or standard chow (controls). Brain phosphagen (i.e., cellular energy reserves) or PCr plus phosphorylated GPA (GPAP) concentrations were equal. The phosphagen-to-NTP ratio was lower than in controls. In vivo CK reaction rate decreased fourfold, whereas ex vivo CK activity that was biochemically measured was doubled. During seizures, CK-catalyzed fluxes increased only in GPA-fed mice. Phosphagen increased in GPA-fed mice, whereas PCr decreased in controls. Survival was higher and brain phosphagen and ATP losses were less for hypoxic GPA-fed mice than for controls. In contrast to mice fed GPA, hypoxic survival and CK reactant concentrations during hypoxia and seizures were the same in creatine-fed mice and controls. Thus GPA, GPAP, or adaptive changes in ATP metabolism stabilize brain ATP and enhance survival during hypoxia in mice.
Intracellular electrolytes regulate the volume set point of the organic osmolyte/anion channel VSOAC
Authors: Emma F. , McManus M. , Strange K.
Published: December 2017

Regulation of the volume sensitivity of the swelling-activated organic osmolyte/anion channel VSOAC by intracellular electrolytes was examined in intact and patch-clamped C6 glioma cells. In intact cells, VSOAC activation was monitored by [3H]taurine efflux measurements, and intracellular electrolyte concentrations were manipulated by acclimation to hypertonic medium for varying periods of time. Hypertonic shrinkage was followed by a rapid and complete regulatory volume increase mediated by electrolyte accumulation that elevated intracellular Na+, K+, and Cl- concentrations. During prolonged (4-48 h) exposure to hypertonicity, electrolyte concentrations decreased gradually as cells accumulated the organic osmolyte myo-inositol. VSOAC activation induced by cell swelling of 35-40% increased as a function of the time of exposure to hypertonicity and was inversely correlated with measured intracellular Na+, K+ and Cl- levels. In patch-clamped cells, swelling-induced Cl- current activation was unaffected by acclimation conditions but was inhibited by increasing the concentration of electrolytes in the patch pipette solution. Quantification of the relationship between VSOAC activation and cell swelling demonstrated that increases in intracellular electrolyte levels increase VSOAC volume set point. Regulation of VSOAC volume sensitivity by electrolytes allows cells to selectively utilize electrolytes or a combination of electrolytes and organic osmolytes for regulatory volume decrease (RVD). Control over the type of solute used for volume regulation is advantageous, allowing cells to control intracellular ionic composition and prevent increases in cytoplasmic ionic strength during RVD.
Cell volume regulation by the mouse zygote: mechanism of recovery from a volume increase
Authors: Seguin D. G. , Baltz J. M.
Published: December 2017

Mouse zygotes regulate their volumes after cell swelling. This regulatory volume decrease (RVD) is rapid and complete. RVD in zygotes was inhibited by K+ or Cl- channel blockers, indicating the participation of such channels in volume recovery. The channels are separate entities, as indicated by the ability of the cation ionophore gramicidin to restore RVD when K+ channels are blocked but not when Cl- channels are blocked. Intracellular Ca2+ concentration increased with cell swelling. Nevertheless, RVD occurred normally in zygotes loaded with the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which prevented Ca2+ from increasing above its normal resting concentration. Thus an increase in intracellular Ca2+ is not necessary for zygote RVD; consistent with this, inhibitors of Ca(2+)-activated K+ channels had little or no effect on RVD. RVD in zygotes was also completely inhibited by millimolar amounts of extracellular ATP. ATP has been shown to inhibit current passed by the volume-sensitive organic osmolyte-Cl- channel in other cells, and thus zygotes may have such a channel participating in RVD.
Role of purinergic receptors in chloride secretion in Caco-2 cells
Authors: Inoue C. N. , Woo J. S. , Schwiebert E. M. , Morita T. , Hanaoka K. , Guggino S. E. , Guggino W. B.
Published: December 2017

Purinergic receptors play an important role in regulating Cl- secretion in epithelial cells. To explore further the role of these receptors in the intestine, we utilized the human intestinal epithelial cell line, Caco-2, grown on permeable membrane supports and assayed for Cl- secretion by measuring the short-circuit current (Isc). Stimulation of Isc by extracellular nucleotides could be detected by day 4 and increased by day 10 postseeding. The magnitude of stimulation of Isc at 10 microM in cells at day 10 was UTP > ATP > UDP > > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) = ADP on the apical side and UTP = 2-MeS-ATP = ATP > ADP > > UDP on the basolateral side. Cross-desensitization studies suggested that two different receptors are expressed in the apical membrane, a P2U purinoceptor and a uridine nucleotide receptor. Two different receptors are also expressed in the basolateral membrane, a P2U receptor and another that reacts with both 2-MeS-ATP and ADP. This latter receptor has an unusual pharmacological profile, with a reactivity for 2-MeS-ATP > ADP but not for ATP. Responses to purinergic receptor agonists were inhibited by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, thapsigargin, or quinine. Thus we suggest that an increase in intracellular Ca2+ and subsequent opening of Ca(2+)-activated K+ channel play a role in increasing driving force for Cl- to exit across the apical membrane. The role of the cystic fibrosis transmembrane conductance regulator as a Cl- exit pathway on the apical membrane was also established.
Whole cell Cl- conductances in mouse choroid plexus epithelial cells do not require CFTR expression
Authors: Kibble J. D. , Garner C. , Colledge W. H. , Brown S. , Kajita H. , Evans M. , Brown P. D.
Published: December 2017

Whole cell patch-clamp studies were performed with tissue isolated from the cystic fibrosis (CF) transgenic Cftrm1cam mouse, to determine whether anion currents in choroid plexus epithelial cells require the expression of cystic fibrosis transmembrane conductance regulator (CFTR). Inclusion of 0.25 mM adenosine 3',5'-cyclic monophosphate (cAMP) and 375 nM protein kinase A (PKA) in the pipette solution caused a significant activation of a Cl(-)-selective, inward-rectifying conductance in cells from wild-type and CF mice. The small, outward currents observed in wild-type and CF animals, however, were not activated by cAMP-PKA. There were no significant differences in the size of currents between wild-type, heterozygote, and CF cells in the presence or absence of cAMP-PKA. A second whole cell conductance was activated when cells from wild-type mice were swollen. These volume-activated currents were Cl- selective and exhibited outward rectification. They were Ca2+ independent and ATP dependent and blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The volume-activated channels were also activated in CF mutant cells, and there was no significant difference in the size of the volume-activated currents between wild-type, heterozygote, and CF cells. It is concluded that CFTR neither contributes to the whole cell conductance nor regulates the other anion conductances in choroid plexus epithelial cells.
Calcium transients and calcium homeostasis in adult mouse fast-twitch skeletal muscle fibers in culture
Authors: Liu Y. , Carroll S. L. , Klein M. G. , Schneider M. F.
Published: December 2017

Skeletal muscle fibers enzymatically dissociated from adult mouse flexor digitorum brevis muscles were maintained in culture for up to 8 days. After various times in culture, fibers were loaded with fura 2, and Ca2+ transients for trains of 1, 5, and 10 action potentials (100 Hz) triggered by external electrical stimulation were calculated from fluorescence ratio records corrected for noninstantaneous reaction of fura 2 with Ca2+. The decay rate constants of Ca2+ transients decreased with increasing stimulation duration, indicating a slowing of the Ca(2+)-removal properties with increased stimulation duration. After 6 days in culture, Ca2+ decay rate constants decreased dramatically for all stimulation durations and the differences in decay rate constants among 1, 5, and 10 pulses became smaller. Intracellular parvalbumin content measured by single-fiber immunofluorescence decreased with time in culture in parallel with the decrease in the decay rate constant of Ca2+ transients. Our results suggest that there is a correlation between parvalbumin content and the decay rate constant of the Ca2+ transient.
Phospholamban ablation enhances relaxation in the murine soleus
Authors: Slack J. P. , Grupp I. L. , Luo W. , Kranias E. G.
Published: December 2017

Phospholamban (PLB) is expressed in slow-twitch skeletal, cardiac, and smooth muscles. Several studies have indicated that it is an important regulator of basal contractility and the stimulatory responses to isoproterenol in the mammalian heart. To determine whether PLB is also a key modulator of slow-twitch skeletal muscle contractility, we examined isometric twitch contractions of isolated, intact soleus muscles from wild-type (WT) and PLB-deficient mice in parallel. Soleus muscles from PLB-deficient mice exhibited a significant (25%) decrease in the time to half relaxation, with no change in contraction time compared with WT soleus muscles. The observed enhancement of relaxation in the PLB-deficient soleus was not associated with alterations in the protein levels of either the sarcoplasmic reticular Ca(2+)-adenosinetriphosphatase or the ryanodine receptor. Examination of the effects of isoproterenol on the twitch kinetics of these muscles revealed 1) no effect on the contraction times of either WT or PLB-deficient muscles and 2) a significant decrease in the half relaxation time of the WT soleus, whereas this parameter remained unchanged in the PLB-deficient muscle. Furthermore, with maximal isoproterenol stimulation, the half relaxation time of the WT soleus was similar to that of the nonstimulated PLB-deficient soleus. These results suggest that PLB is a key determinant of relaxation in slow-twitch skeletal muscle under basal conditions and during isoproterenol stimulation.
Ca(2+)-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle
Authors: Heppner T. J. , Bonev A. D. , Nelson M. T.
Published: December 2017

The goal of this study was to examine the role of large conductance Ca(2+)-activated K+ channels in the regulation of cell excitability in urinary bladder smooth muscle from the guinea pig. Ca(2+)-activated K+ channels were studied with single-channel recording techniques and found to be intracellular Ca2+ and voltage dependent and sensitive to external tetraethylammonium and blocked by nanomolar concentrations of iberiotoxin (apparent dissociation constant of 4 nM). Spontaneous action potentials recorded from intact tissue strips depended on external Ca2+ and were inhibited by Ca2+ channel blockers. Iberiotoxin (100 nM) significantly altered the configuration of the action potential by increasing the duration and peak amplitude of the action potential and decreasing the rate of decay. Iberiotoxin also increased the action potential frequency from 0.11 to 0.29 Hz. This study suggests that Ca(2+)-activated K+ channels play a significant role in the repolarization of the action potential and in the maintenance of the resting membrane potential of the urinary bladder smooth muscle.
Characterization of volume-sensitive taurine- and Cl(-)-permeable channels
Authors: Galietta L. J. , Falzoni S. , Di Virgilio F. , Romeo G. , Zegarra-Moran O.
Published: December 2017

Volume-sensitive Cl- channels [ICl(vol)] were studied using taurine efflux and patch-clamp experiments in 9HTEo- human tracheal cells. Cells were stimulated with the Ca(2+)- elevating agents ATP and ionomycin in isotonic medium or in hypotonic solutions. ATP (100 microM) or ionomycin (1 microM) and hypotonic shock produced a synergic effect. Indeed, the resulting taurine efflux was much higher than the sum of the single effects elicited by ATP, ionomycin, or hypotonic medium. The taurine release elicited by hypotonic shock and the potentiation by ATP and ionomycin were markedly inhibited by using a Ca(2+)-free extracellular medium and by incubating the cells with the membrane-permeable 1,2-bis(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester chelating agent. Patch-clamp experiments confirmed the role of Ca2+ on ICl(vol) channels. Swelling-induced taurine efflux was inhibited by reactive blue 2, suramin, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Patch-clamp experiments demonstrated that these compounds shift the voltage-dependent inactivation of ICl(vol) channels toward more negative values. This study indicates that the sensitivity of ICl(vol) to cell volume changes is modulated by intracellular Ca2+ and that purinergic receptor antagonists represent a new class of CI- channel blockers.
Exogenous fructose 1,6-bisphosphate reduces K+ permeability in isolated rat hepatocytes
Authors: Roig T. , Bartrons R. , Bermudez J.
Published: December 2017

The relationship between the protective effect of fructose 1,6-bisphosphate (F-1,6-P2) against cell injury and the modifications produced in the metabolic fluxes and in the membrane permeability to K+ was studied in isolated rat hepatocytes. Incubation of these cells in the presence of F-1,6-P2 reduced metabolic activity without affecting the ATP content, which suggests a downregulation of the ATP turnover. Using 86Rb+ as a tracer, we analyzed the relationship between these metabolic changes and alterations in K+ fluxes. In the presence of F-1,6-P2 the passive and the active K+ fluxes in hepatocytes decreased. However, the Na(+)-K+ pump from semipurified membranes was not directly affected by F-1,6-P2, which suggests a secondarily induced reduction of Na(+)-K+ pump activity. Moreover, galactosamine-treated cells showed a marked increase in permeability to K+ that was abolished by the presence of F-1,6-P2. This protective effect may be related to the prevention of K+ efflux. The results reported here strongly suggest the induction of channel arrest, and the associated metabolic downregulation, as the primary protective effect of F-1,6-P2, as has been shown in the prevention of galactosamine-induced hepatotoxicity.
Ca(2+)-activated Cl- currents are activated by metabolic inhibition in rat pulmonary artery smooth muscle cells
Authors: Wang Q. , Wang Y. X. , Yu M. , Kotlikoff M. I.
Published: December 2017

We report the electrophysiological and functional properties of Ca(2+)-activated Cl- currents [ICl(Ca)] in rat pulmonary artery smooth muscle and the activation of these currents by the metabolic inhibitor cyanide. Caffeine and norepinephrine (NE) evoked both Ca(2+)-activated K+ currents [IK(Ca)] and ICl(Ca) currents in voltage-clamped myocytes (-50 mV). Niflumic acid (10 microM) reduced the caffeine-induced ICl(Ca) by approximately 64% and reversibly reduced NE-induced tension. Exposure of myocytes to cyanide (2-10 mM) induced a slowly developing inward current (-50 mV) in physiological and K(+)-free solutions, which was identified as ICl(Ca) on the basis of ion selectivity and Ca2+ dependence. Cyanide elevated cytosolic Ca2+ concentration, and this elevation was markedly inhibited by preexposure to caffeine and slightly inhibited by nisoldipine. During exposure to caffeine, the Ca(2+)-activated K+ current was also augmented. Cyanide markedly prolonged ICl(Ca) activated by caffeine, increasing the half-decay time from 3.5 (control) to 29 s (cyanide); the half-decay time of the caffeine-induced IK(Ca) was not significantly affected by cyanide. The results indicate that metabolic inhibition increases [Ca2+]i and activates a prolonged, depolarizing Cl- current in pulmonary artery myocytes.
Polyamines modulate transcription but not posttranscription of c-myc and c-jun in IEC-6 cells
Authors: Patel A. R. , Wang J. Y.
Published: December 2017

The goal of the current study was to examine whether polyamines are involved in the regulation of transcription and posttranscription of the protooncogenes c-myc and c-jun in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis, for 4 or 6 days not only almost completely depleted total (whole) cellular and nuclear polyamines but also significantly decreased expression of the protooncogenes c-myc and c-jun in IEC-6 cells. Using nuclear run-on transcription assay, we demonstrated that the basal rate of transcription of c-myc was decreased by 55% at 4 days and by 60% at 6 days in the DFMO-treated cells. The c-jun transcription in DFMO-treated cells was decreased by 75% at 4 days and 85% at 6 days. The transcription rates of c-myc and c-jun were dramatically stimulated by 5% dialyzed fetal bovine serum (dFBS) in normal quiescent cells. However, polyamine depletion significantly prevented the increased transcription of these two genes in the DFMO-treated cells exposed to 5% dFBS. Furthermore, direct administration of spermidine to isolated nuclei from polyamine-deficient (caused by DFMO) cells resulted in a 2- to 2.5-fold increase in c-myc and c-jun transcription. There were no significant changes in the half-lives of c-myc and c-jun mRNAs between the controls and the DFMO-treated cells. These results indicate that 1) polyamines are required for the transcription of the protooncogenes c-myc and c-jun in IEC-6 cells and 2) depletion of intracellular polyamines has no effect on posttranscriptional regulation of c-myc and c-jun mRNAs. These findings suggest that polyamines play an important role in the regulation of the transcription of protooncogenes, and this may be one mechanism by which polyamines modulate mucosal cell division.
Single-fiber and whole muscle analyses of MHC isoform plasticity: interaction between T3 and unloading
Authors: Caiozzo V. J. , Baker M. J. , McCue S. A. , Baldwin K. M.
Published: December 2017

Previous data suggest that separate interventions of hyperthyroidism (T3) and hindlimb suspension (HS) act on some but not all slow type I fibers in the soleus muscle. This may be due to the presence of "refractory" fibers that are unresponsive to either of these interventions. Alternatively, T3 and HS might act on different populations of slow type I fibers in the soleus muscle. Adult female Sprague-Dawley rats were assigned to 1) control, 2) T3, 3) HS, or 4) T3+HS. Nine animals were assigned to each group. Single-fiber electrophoretic analyses (n = 40 per muscle) of the soleus muscle demonstrated that the HS reduced the percentage of slow type I fibers from approximately 80% (control) to approximately 40% (HS) of the fiber population. Although hyperthyroidism affected a greater percentage of slow type I fibers than HS, a small population (approximately 10% of the slow type I fibers) were unaffected by T3. The combined intervention, in contrast, transformed all slow type I fibers into fibers expressing various combinations of fast myosin heavy chain (MHC) isoforms. These findings demonstrate that the soleus muscle does not contain so-called refractory fibers. They further suggest that the soleus muscle contains different populations of slow type I fibers that vary in their sensitivity to altered physiological conditions.
Intestinal ischemia and reperfusion injury in transgenic mice overexpressing copper-zinc superoxide dismutase
Authors: Deshmukh Devendra R. , Mirochnitchenko Oleg , Ghole Vikram S. , Agnese Doreen , Shah Pritesh C. , Reddell Michael , Brolin Robert E. , Inouye Masayori
Published: December 2017

Superoxide dismutase (SOD) scavenges oxygen radicals that are implicated in the pathogenesis of intestinal ischemia-reperfusion injury. The effect of intestinal ischemia and reperfusion was investigated in transgenic mice overexpressing human Cu-Zn SOD. Ischemia was induced by occluding the superior mesenteric artery. Myeloperoxidase activity was determined as an index of neutrophil infiltration, and malondialdehyde levels were measured as an indicator of lipid peroxidation. Forty-five minutes of intestinal ischemia followed by 4 h of reperfusion caused an increase in intestinal levels of malondialdehyde in both nontransgenic and transgenic mice, but the concentration of malondialdehyde was significantly greater in nontransgenic mice. Intestinal ischemia-reperfusion also caused an increase in intestinal and pulmonary myeloperoxidase activity in nontransgenic and transgenic mice, but the transgenic mice had significantly lower levels of myeloperoxidase activity than nontransgenic mice. Transgenic mice had higher levels of intestinal SOD activity than nontransgenic mice. There were no significant differences in the catalase or glutathione peroxidase activities. In conclusion, our study demonstrates that the overexpression of SOD protects tissues from neutrophil infiltration and lipid peroxidation during intestinal ischemia-reperfusion.
Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis
Authors: Ishihara Hiroshi , Welsh Michael J.
Published: December 2017

ATP hydrolysis by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel predicts that energy from hydrolysis might cause asymmetric transitions in the gating cycle. We found that 3-( N-morpholino)propanesulfonic acid (MOPS) blocked the open channel by binding to a site 50% of the way through the electrical field. Block by MOPS revealed two distinct states, O1 and O2, which showed a strong asymmetry during bursts of activity; the first opening in a burst was in the O1 state and the last was in the O2 state. Addition of a nonhydrolyzable nucleoside triphosphate prevented the transition to the O2 state and prolonged the O1 state. These data indicate that ATP hydrolysis by the nucleotide-binding domains drives a series of asymmetric transitions in the gating cycle. They also indicate that ATP hydrolysis changes the conformation of the pore, thereby altering MOPS binding.
β-Adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes
Authors: Kanai Anthony J. , Mesaros Stephan , Finkel Mitchell S. , Oddis Carmine V. , Birder Lori A. , Malinski Tadeusz
Published: December 2017

Nitric oxide (NO) has been implicated in endogenous control of myocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surface of individual neonatal or adult rat ventricular myocytes ( n > 6 neonatal and adult cells per experiment). In beating neonatal myocytes, there was no detectable spontaneous NO release with each contraction. However, norepinephrine (NE; 0.25–1 μM) elicited transient NO release from beating neonatal (149 ± 11 to 767 ± 83 nM NO) and noncontracting adult (157 ± 13 to 791 ± 89 nM NO) cells. NO was released by adrenergic agonists with the following rank order of potency: isoproterenol (β1β2) > NE (α/β1) > dobutamine (β1) ≈ epinephrine (α/β1β2) > tertbutylene (β2); NO was not released by phenylephrine (α). NE-evoked NO release was reversibly blocked by N G-monomethyl-l-arginine, trifluoperazine, guanosine 5′- O-(2-thiodiphosphate), and nifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 ± 1.6%) and BAY K 8644 (10 μM = 11.9 ± 1%). NO was also released by A-23187 (10 μM = 884 ± 88 nM NO), guanosine 5′- O-(3-thiotriphosphate) (1 μM = 334 ± 56 nM NO), and dibutyryl adenosine 3′,5′-cyclic monophosphate (10–100 μM = 35 ± 9 to 284 ± 49 nM NO) but not by ATP, bradykinin, carbachol, 8-bromoguanosine 3′,5′-cyclic monophosphate, or shear stress. This first functional demonstration of a constitutive NO synthase in cardiac myocytes suggests its regulation by a β-adrenergic signaling pathway and may provide a novel mechanism for the coronary artery vasodilatation and enhanced diastolic relaxation observed with adrenergic stimulation.
Kinetics of creatine kinase in an experimental model of low phosphocreatine and ATP in the normoxic heart
Authors: Stepanov V. , Mateo P. , Gillet B. , Beloeil J. C. , Lechene P. , Hoerter J. A.
Published: December 2017

To study the dependence of the forward flux of creatine kinase (CK) on its substrates and products we designed an acute normoxic model of steady-state depletion of phosphocreatine (PCr) and adenylate in the isovolumic acetate-perfused rat heart. Various concentrations of PCr and ATP were induced by prior perfusion with 2 deoxy-d-glucose in the presence of insulin. The apparent rate constant ( k f) and the forward CK flux were measured under metabolic and contractile steady state by progressive saturation-transfer31P nuclear magnetic resonance (NMR). At high adenylate content CK flux was constant for a twofold reduction in PCr concentration ([PCr]); CK flux was 6.3 ± 0.6 mM/s (vs. 6.5 ± 0.2 mM/s in control) because of a doubling of k f. Although, at the lowest ATP concentration and [PCr], CK flux was reduced by 50%, it nevertheless always remained higher than ATP synthesis estimated by parallel oxygen consumption measurement. NMR-measured flux was compared with the flux computed under the hypothesis of CK equilibrium. CK flux could not be fully predicted by the concentrations of CK metabolites. This is discussed in terms of metabolite and CK isozyme compartmentation.