bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional expression of the rat anion exchanger AE2 in insect cells by a recombinant baculovirus
Authors: He X. , Wu X. , Knauf P. A. , Tabak L. A. , Melvin J. E.
Published: December 2017

We used baculovirus to transiently express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. No detectable Cl(-)-HCO3- exchange activity was observed in wild type or sham-infected Sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a pH-sensitive fluorescent dye. Functional expression of anion exchange activity in the AE2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. The expressed AE2 anion exchange activity was Na+ independent and could be reversibly and irreversibly inhibited by the specific anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The reversible inhibition was sensitive to the concentration of DIDS, with a half inhibition of 4 microM. These results indicate that the rat AE2 protein produced in the recombinant baculovirus-infected insect cells is inserted into the plasma membrane in a biologically active form that appears suitable for functional studies of AE2.
Phosphorylation of Na(+)-H+ antiporter is not stimulated by phorbol ester and acidification in granulocytic HL-60 cells
Authors: Rao G. N. , Sardet C. , Pouyssegur J. , Berk B. C.
Published: December 2017

During differentiation of HL-60 cells into granulocyte-like cells, mRNA and protein levels for the Na(+)-H+ antiporter increased 10- to 15-fold. However, functional activity, as measured by recovery from an acid load [intracellular pH (pHi) 6.5] increased by only about twofold. In addition, basal pHi (measured in the absence of bicarbonate) increased from 7.15 to 7.26, suggesting an alteration in the antiporter's "set point" during HL-60 cell differentiation. To gain insight into the role of the Na(+)-H+ antiporter in HL-60 cell differentiation, we studied mRNA expression of the NHE-1, NHE-3, and NHE-4 isoforms. Only the NHE-1 isoform mRNA increased during differentiation. Because it has recently been shown that the antiporter is regulated by phosphorylation, we next studied NHE-1 protein phosphorylation during HL-60 cell differentiation. Differentiation by exposure to 1 microM retinoic acid for 6 days caused a 15-fold increase in the synthesis of the NHE-1 protein. However, immunoprecipitation of 32P-labeled antiporter showed a decrease in band intensity. These data indicate that during HL-60 cell differentiation, there was a net decrease in the phosphorylation of NHE-1 despite an increase in pHi. Nonetheless, recovery from an acid load (pHi 6.51) was significantly more rapid in differentiated than control cells: 62 +/- 6 vs. 38 +/- 8 mmol H+.min-1.1 cells-1, respectively. However, acid loading decreased antiporter phosphorylation by twofold in differentiated and undifferentiated HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Properties of whole cell currents in isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera
Authors: Delgado R. , Labarca P.
Published: December 2017

Isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera were found to possess a same set of currents. Outward currents, made of a delayed rectifier and a Ca(2+)-dependent component, were blocked by replacing K+ by Cs+ in the patch pipette, in the presence of millimolar concentrations of tetraethylammonium and 4-aminopyridine in the external solution. Inward currents were made of a transient and a maintained component. The transient was abolished in the absence of external Na+ and was blocked by tetrodotoxin, with an apparent dissociation constant (KDapp) of 25.4 +/- 0.3 nM. The maintained inward currents were suppressed on removing external Ca2+, could be carried also by Ba2+, and were selectively blocked by Cd2+ (KDapp = 3.2 +/- 1.3 microM). A variety of agents found to block the maintained Ca2+ inward currents, including Co2+ and Ni2+, at millimolar concentrations, and nifedipine, verapamil, amiloride, and the amiloride analogue benzamil, at micromolar concentrations, were also effective in either modifying the gating of, or in blocking, the transient inward currents.
Expression of myosin regulatory light-chain isoforms and regulation of phosphorylation in smooth muscle
Authors: Monical P. L. , Owens G. K. , Murphy R. A.
Published: December 2017

Our objectives were to 1) determine how growth state and cell density affect the expression of the smooth muscle (SM) and nonmuscle (NM) isoforms of the 20-kDa myosin regulatory light chains (MLC20) in cultured rat aortic smooth muscle cells (SMC) and 2) to determine whether angiotensin II stimulates differential phosphorylation of SM and NM MLC20 isoforms in an effort to assess whether the SM and NM isoforms may subserve different cellular functions. The results demonstrated that changes in the SM MLC20 isoform content were inversely correlated with cell growth but independent of cell density. MLC20 phosphorylation levels were 20.8 +/- 2.9 and 19.4 +/- 3.7% for SM and NM isoforms, respectively, in unstimulated, substrate-attached SMC. Angiotensin II transiently elevated phosphorylation levels of both the SM and NM MLC20 isoforms to 60-70%. No differences in either the magnitude or the kinetics of phosphorylation were observed for the SM vs. NM isoforms. Forskolin, 3-isobutyl-1-methylxanthine, or isoproterenol treatment led to parallel dephosphorylation of the SM- and NM-specific isoforms followed by depolymerization of stress fibers and cell arborization. The studies provide evidence that growth arrest of cultured SMC enhances expression of cell-specific/-selective proteins characteristic of differentiated SM. However, there was no evidence for differential phosphorylation changes of SM and NM MLC20 isoforms in response to activating or relaxing agents as expected if these isoforms subserve different cellular functions.
Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues
Authors: Gatto C. , Milanick M. A.
Published: December 2017

This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.
Tetramethylammonium activation of muscarinic receptors in cardiac ventricular myocytes
Authors: Zakharov S. I. , Overholt J. L. , Wagner R. A. , Harvey R. D.
Published: December 2017

Replacement of extracellular Na+ with tetramethylammonium (TMA) reduces the magnitude of the Cl- current activated by beta-adrenergic receptor stimulation in guinea pig ventricular myocytes. However, the effects of replacing Na+ appear to be associated with the presence of TMA, rather than the absence of Na+. Direct addition of TMA to extracellular solutions, without changing the Na+ concentration, was able to inhibit the Cl- current activated by isoproterenol (Iso) in a concentration-dependent manner. The concentration of TMA that caused half-maximal inhibition was 327 microM when the Cl- current was activated by 1 microM Iso and 29 microM when the Cl- current was activated by 0.03 microM Iso. The effect of TMA was also blocked by atropine, suggesting that TMA exerts its effect through stimulation of the muscarinic receptors. Furthermore, TMA inhibited the Iso-activated Ca2+ current, as would be expected for an effect involving muscarinic receptor stimulation. The response to complete Na+ replacement with TMA could not be overcome by increasing the concentration of Iso 1,000-fold, and direct addition of TMA was able to antagonize the Cl- current activated independently of the beta-adrenergic receptor, using forskolin and histamine. These results are consistent with the hypothesis that TMA does not exert its effects through a competitive mechanism at the beta-adrenergic receptor. It is concluded that TMA is able to antagonize adenosine 3',5'-cyclic monophosphate-dependent activation of ion channels in the heart through activation of muscarinic receptors.
Comparison of Na+ currents from type IIa and IIb human intercostal muscle fibers
Authors: Ruff R. L. , Whittlesey D.
Published: December 2017

The voltage dependence and amplitude of Na+ currents (INa) were studied with the loose-patch voltage-clamp technique on 19 fast-twitch human intercostal skeletal muscle fibers at the endplate border and > 200 microns from the endplate (extrajunctional). The fibers were histochemically classified as fast-twitch oxidative-glycolytic (type IIa, n = 9) or fast-twitch glycolytic (type IIb, n = 10). The voltage dependence of activation and fast and slow inactivation of INa were similar for membrane patches recorded on the endplate border and on extrajunctional membrane for both fiber types. INa was about fivefold larger on the endplate border compared with extrajunctional membrane for both fiber types. Type IIb fibers had larger values of INa and manifest fast inactivation of INa at more negative potentials than type IIa fibers. The difference between type IIa and IIb fibers may enable IIb fibers to operate at higher firing frequencies for brief periods.
Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric
Authors: Luo H. , Tesfaye A. , Schieren I. , Chase H. S.
Published: December 2017

Madin-Darby canine kidney (MDCK) cells were transfected with the cDNA for the rat 5-HT1C receptor (pMV7-SR1c) using electroporation. Cells that survived G418 selection medium were loaded with indo-1 and run through a fluorescence-activated cell sorter (FACS); 10% responded to serotonin (5-HT) with an increase in intracellular Ca2+ concentration ([Ca2+]i). Responding cells were separated with the FACS, grown to confluence, and resorted two more times until a clone of 100% respondents was obtained (SR-MDCK). In SR-MDCK cells grown on porous filters, [Ca2+]i increased only when 5-HT was applied to the basolateral membrane (change in [Ca2+]i = 190 +/- 43 nM); there was no response of [Ca2+]i to apical application of 5-HT. The asymmetric response to 5-HT was likely due to targeting of 5-HT1C receptors exclusively to the basolateral membrane of SR-MDCK cells; 125I-labeled lysergic acid diethylamide binding sites, a marker of high-affinity 5-HT receptors, were located only in the basolateral membrane. These experiments demonstrate that epithelial cells can be stably transfected to express G protein-linked, calcium-mobilizing receptors and that the receptors may be targeted asymmetrically to specific domains of the plasma membrane.
Insulin-induced membrane changes in K(+)-depleted rat skeletal muscle
Authors: Bond E. F. , Gordon A. M.
Published: December 2017

Insulin-induced membrane changes were investigated in K(+)-depleted rat muscle. Male Sprague-Dawley rats were placed on a K(+)-free but otherwise adequate diet for 5-8 wk; serum K+ concentration ([K+]) dropped to 1.2-3.2 mM. Omohyoid membrane potential was -81 mV in 5.5 mM [K+] (SO4(2-)). Exposure to either insulin or low (0.5 mM) [K+] singly changed potential only slightly. The combination resulted in depolarization of 90% of fibers (-43 mV) and hyperpolarization of 10% of fibers (-101 mV). Fibers from normokalemic rats did not depolarize. Tetrodotoxin (TTX) blocked depolarization, implying the presence of noninactivating TTX-sensitive Na+ channels. K+ currents were measured using the three-electrode voltage clamp; movement of other ions was prevented by ion substitution, channel blockers, and depolarization-induced channel inactivation. K+ conductance was similar in control fibers with or without insulin. In the absence of insulin, currents in K(+)-depleted fibers were offset by a large leakage current that was significantly diminished when insulin was present. The insulin-induced current decrease was observed in nitrendipine, suggesting that the apparent decreased outward current was not an inward current carried by Ca2+. Data are consistent with altered Na+ and K+ channels in K(+)-depleted muscle, i.e., insulin-related closing of K+ channels initiates depolarization, which is then sustained by opening of noninactivating Na+ channels.
Proton conductance of the plasma membrane: properties, regulation, and functional role
Authors: Lukacs G. L. , Kapus A. , Nanda A. , Romanek R. , Grinstein S.
Published: December 2017

H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in the case of phagocytes. When stimulated, these cells undergo a sustained depolarization, while generating large amounts of metabolic acid. In addition, conductive H+ fluxes may also provide counterions to neutralize the activity of electrogenic enzymes, as suggested for the phagocyte NADPH oxidase.
Albumin and Ricinus communis agglutinin decrease endothelial permeability via interactions with matrix
Authors: Qiao R. , Siflinger-Birnboim A. , Lum H. , Tiruppathi C. , Malik A. B.
Published: December 2017

We studied the effects of albumin and the lectin Ricinus communis agglutinin (RCA) on hydraulic conductivity (Lp) of bovine pulmonary microvascular endothelial cell monolayers (BPMVEC) because of the evidence that albumin and RCA can interfere with transendothelial albumin permeability (Siflinger-Birnboim, A., J. Schnitzer, H. Lum, F. Blumenstock, C. Shen, P. Del Vecchio, and A. Malik. J. Cell. Physiol. 149: 575-584, 1991). BPMVEC were seeded on microporous polycarbonate filters, and the liquid flux was measured by collecting effluent into a tubing of known inner diameter at transendothelial hydrostatic pressures (P) ranging from 5 to 20 cmH2O. Lp was calculated as the slope of the relationship of liquid flux per unit surface area (Jv) vs. P. Addition of RCA (50 micrograms/ml) or albumin (5 mg/ml) to the endothelial cell medium containing albumin-free Hanks' balanced saline solution (HBSS) decreased total Lp (expressed x 10(-6) cm.s-1 x cmH2O-1) from 17.2 +/- 3.6 during HBSS to 4.7 +/- 0.9 during albumin and 5.7 +/- 1.6 during RCA (P < 0.01 for both). The RCA effect, but not that of albumin, was prevented by the addition of D-galactose (0.1 M) (the cognate hapten monosaccharide of RCA). We determined the contribution of the extracellular matrix (ECM) in decreasing the Lp by obtaining ECM after treatment of the monolayers with 0.025 M NH4OH to detach endothelial cells from the ECM. Basal ECM Lp (expressed x 10(-6) cm.s-1 x cmH2O-1) was 57.0 +/- 15.3, and it decreased to 19.7 +/- 4.3 and 17.5 +/- 2.9 during RCA and albumin, respectively (P < 0.01 for both). In contrast, RCA and albumin did not alter the filter Lp values. Another lectin, Ulex europaeus agglutinin, and the protein immunoglobulin G had no effect on Lp values.(ABSTRACT TRUNCATED AT 250 WORDS)
Insulin uptake and processing by cultured mouse glomerular endothelial cells
Authors: Rabkin R. , Tsao T. , Elliot S. J. , Striker L. J. , Striker G. E.
Published: December 2017

Endothelial cells isolated from a variety of vascular beds bind and transport insulin but exhibit relatively low insulin degrading activity. Because endothelial cells exhibit heterogeneity and since kidney is a major site of insulin degradation, we studied the processing of insulin by glomerular endothelial cells (GEC). When exposed to 2 x 10(-10) M 125I-labeled insulin, GEC associated with the hormone in a specific manner. This interaction was inhibited by insulin but not by a number of unrelated peptide hormones. Over a 90-min period, GEC degraded 42 +/- 3% of the 125I-insulin, as measured by solubility in trichloroacetic acid (TCA). Degradation was inhibited 90% by an excess of insulin or adrenocorticotropic hormone (10(-6) M) and 57% by glucagon, whereas growth hormone and calcitonin were without effect. Separation of plasma membrane bound from internalized insulin was achieved by decreasing extracellular pH. In the steady state, 43% of cell-associated insulin was membrane bound and 57% internalized. The fate of the internalized 125I-insulin was examined by incubating acid-washed cells at 37 degrees C for 60 min. Over this time 18% of the radioactivity was released as TCA insoluble- and 72% as TCA-soluble radioactivity. Release was increased by insulin (10(-6) M) but not by unrelated peptide hormones. In the presence of chloroquine, 125I-insulin release increased by one third while degradation fell. High-performance liquid chromatography revealed that GEC released both intact insulin and large intermediates and that chloroquine inhibited intermediate formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes
Authors: Dubinsky W. P. , Mayorga-Wark O. , Garretson L. T. , Schultz S. G.
Published: December 2017

We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.
Further characterization of the Na-Ca exchange-dependent Ca compartment in rat ventricular cells
Authors: Langer G. A. , Rich T. L.
Published: December 2017

A discrete subcellular Ca compartment, absolutely dependent upon Na-Ca exchange for its exchange, has been previously identified in ventricular cells from both adult rat and from neonatal rat cultures. The present study further characterizes this compartment in the adult ventricular myocyte. The compartment's exchange is significantly decreased by 20 microM 3,4-dichlorobenzamil. Its content responds significantly to variation of the extracellular Ca concentration ([Ca]o) and to reversal of the Na-Ca exchanger by reduction of extracellular Na concentration to zero. Its content decreases to 155 +/- 24 mumol/kg dry cell wt in 0.2 mM [Ca]o and increases to 623 +/- 48 mumol/kg at 6.5 mM [Ca]o, where it appears to be near saturation. The compartment is significantly reduced by 45Ca labeling in the presence of 10 mM caffeine (23%) and 1 mM ryanodine (22%). This compartment is not significantly affected by 1 microM thapsigargin. 45Ca activity is retained in the compartment when 45Ca washout is conducted without Na or Ca in the washout solution, i.e., Na-Ca exchange is not operative. Under these conditions, addition of 1 mM Cd to the washout produces a significant release of 45Ca from the compartment. The results are interpreted to indicate that the compartment in the adult cell has two components that are in equilibrium: a relatively small sarcoplasmic reticulum (SR) component and a larger "non-SR" component. This is in contrast to the compartment in the cultured cell in which the compartment is 2.6 times larger and has a larger SR component. The possibility that the non-SR component represents Ca bound to the cytoplasmic leaflet of the sarcolemma at SR junction regions is discussed.
Cl- secretion by cultured shark rectal gland cells. III. Ca2+ regulation of apical membrane Cl- conductance
Authors: Moran W. M. , Valentich J. D.
Published: December 2017

Calcium ionophores (ionomycin and A-23187) were employed to assess the effects of increased intracellular Ca2+ concentration ([Ca2+]i) on apical membrane Cl- conductance (GaCl) and rate of transepithelial Cl- secretion in cultured shark rectal gland (SRG) cells. Apical 2 microM ionomycin induced dramatic changes in cellular electrophysiological properties: the apical membrane electrical potential difference (V(a)) depolarized from -66 mV to -46 mV, the fractional resistance of the apical membrane (fRa) decreased from 0.88 to 0.23, and the transepithelial electrical potential difference (Vab) increased slightly from +1.2 mV to +1.4 mV. These effects result from increased GaCl because apical low-Cl- shark Ringer (SR) depolarized V(a) by 32 mV and increased fRa from 0.23 to 0.36. Ionomycin-stimulated Vab or short-circuit current (Isc) results largely from increased Cl- secretion because approximately 80% of the increase in Isc is Cl- dependent. Establishing the Ca2+ dependence of ionophore activation of GaCl was confounded because apical low-Ca2+ SR [Ca2+ concentration ([Ca2+]) 1 mM to 0.1 microM] alone activated this conductive pathway. To establish the Ca2+ dependence of ionophore action, we assessed the effect of ionomycin on Isc in low-Ca2+ SR ([Ca2+] = 0.1 microM) and in SR. In low-Ca2+ SR, apical ionomycin stimulated Isc by 14.0 microA/cm2. In SR (normal [Ca2+]), ionomycin increased Isc further by 27.0 microA/cm2. Superfusing the basolateral surface with 2 microM ionomycin for 8-16 min failed to activate GaCl. In every case, subsequent superfusion of the apical surface with ionophore for 1.5-2 min activated GaCl. Bilateral 4 microM indomethacin (45-min superfusion) failed to block the ionomycin-induced GaCl.(ABSTRACT TRUNCATED AT 250 WORDS)
Uptake and caffeine-induced release of calcium in fast muscle fibers of Xenopus laevis: effects of MgATP and P(i)
Authors: Stienen G. J. , van Graas I. A. , Elzinga G.
Published: December 2017

To elucidate the origin of the reduction in force during prolonged muscle fatigue, the dependency of Ca2+ uptake and release on MgATP and P(i) concentration was studied in saponin-skinned fast skeletal muscle fibers of the iliofibularis muscle of Xenopus laevis at 3 degrees C. The sarcoplasmic reticulum was loaded with Ca2+ for 5 min at pCa 7.0. The amount of Ca2+ released was derived from the area of the caffeine-induced force response. Ca2+ uptake increased with the MgATP concentration present during loading. It was half maximal at 20 microM and saturated at higher concentrations. The kinetics of Ca2+ release were affected for MgATP concentrations between 0.1 and 0.5 mM or less, but the amount of Ca2+ released by caffeine in ATP-free solutions was substantial. Phosphate (15 mM) only slightly reduced Ca2+ uptake when the loading period was short (1 min). It is unlikely, therefore, that the reduction in MgATP concentration contributes to the depression of Ca2+ released from the sarcoplasmic reticulum during fatigue. The increase in P(i) concentration could play a small role by reducing Ca2+ uptake.
Thyroid hormone specifically regulates skeletal muscle Na(+)-K(+)-ATPase alpha 2- and beta 2-isoforms
Authors: Azuma K. K. , Hensley C. B. , Tang M. J. , McDonough A. A.
Published: December 2017

The purpose of this study was to determine the pattern of thyroid hormone (triiodothyronine, T3) regulation of the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) alpha- and beta-subunit expression in skeletal muscle, which expresses alpha 1-, alpha 2-, beta 1-, and beta 2-subunits, and compare it with that seen in kidney, which expresses only alpha 1 and beta 1. Three steady states were studied: hypothyroid, euthyroid, and hyperthyroid (hypothyroids injected daily with 1 microgram T3/g body wt for 2-16 days). Protein and mRNA abundance, determined by Western and Northern analysis, were normalized to a constant amount of homogenate protein and total RNA, respectively. In skeletal muscle, there was no change in alpha 1- or beta 1-mRNA or protein levels in the transition from hypothyroid to hyperthyroid. However, alpha 2 was highly regulated; mRNA reached a new steady-state level of fivefold over hypothyroid by 8 days of T3 treatment and protein abundance increased threefold. In addition, beta 2-mRNA and protein were detected in skeletal muscle and were also highly regulated by T3; beta 2-mRNA increased nearly fourfold over hypothyroid level, and beta 2-protein abundance increased over twofold. In kidney in the transition from hypothyroid to hyperthyroid, there were coordinate 1.6-fold increases in both alpha 1- and beta 1-mRNA abundance that predicted the observed changes in alpha 1- and beta 1-protein levels and Na(+)-K(+)-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Histamine stimulates a biphasic calcium response in the human tracheal epithelial cell line CF/T43
Authors: Harris R. A. , Hanrahan J. W.
Published: December 2017

We have studied the cytosolic free Ca2+ concentration ([Ca2+]i) response to histamine and other inflammatory mediators in a cystic fibrosis tracheal epithelial cell line (CF/T43) using digital fluorescence imaging. Brief pulses of histamine increased [Ca2+]i in a concentration-dependent manner with threshold, half-maximal, and maximal responses at approximately 5 microM, 120 microM, and 10 mM, respectively. The calcium response to sustained histamine exposure was markedly biphasic, consisting of an early peak (to approximately 2.4 microM [Ca2+]i) followed by a smaller second peak that lasted 45-60 s. Neither peak was directly dependent on Ca2+ influx. In contrast, stimulation with bradykinin gave a single peak followed by a smooth decay back to baseline levels. Sustained perfusion with histamine did not affect the bradykinin response, which is known to be mediated by inositol 1,4,5-trisphosphate (IP3). The H1-type histamine receptor blockers mepyramine, diphenhydramine, and (+)-chlorpheniramine were potent antagonists of the histamine response. Diphenhydramine was also a weak agonist at high concentrations (> or = 1 mM) and gave a biphasic response similar to that with histamine. The H2-type receptor blocker cimetidine and the H3-type receptor blocker thioperamide had no effect. Indomethacin failed to inhibit the second phase of the histamine-stimulated [Ca2+]i response, suggesting that the second [Ca2+]i peak is not due to secondary production of prostaglandins. Neomycin, which inhibits IP3 production, completely abolished the [Ca2+]i response to bradykinin stimulation but did not affect the second phase of the histamine response. The biphasic nature of the histamine response, the insensitivity of the second [Ca2+]i peak to neomycin, and the independence of bradykinin and histamine responses suggest that histamine may modulate [Ca2+]i through multiple IP3 and non-IP3 pathways.
Single fiber analyses of type IIA myosin heavy chain distribution in hyper- and hypothyroid soleus
Authors: Caiozzo V. J. , Swoap S. , Tao M. , Menzel D. , Baldwin K. M.
Published: December 2017

The objectives of this study were to 1) examine the effect of hypo- and hyperthyroidism (triiodothyronine treatment) on the distribution of type IIA myosin heavy chain (MHC) in the soleus at the single fiber level and 2) correlate changes in the single fiber distribution of type IIA MHC with the maximal shortening velocity of whole skeletal muscle. The presence of the type IIA MHC in single fibers was determined using a monoclonal antibody reactive to the type IIA MHC and quantified with a Meridian ACAS 570 interactive laser cytometer. The findings of this study demonstrate that 1) hyperthyroidism significantly increases the relative number of muscle fibers that express type IIA MHC, 2) not all type I fibers are capable of expressing fast type IIA MHC under hyperthyroid conditions, and 3) there is a high correlation between maximal shortening velocity and the relative number of type IIA fibers. This latter observation suggests that the maximal shortening velocity of whole skeletal muscle may not be solely determined by its fastest fiber(s) but rather by the relative proportion of fibers expressing fast type IIA MHC.
Phorbol ester sequentially downregulates cAMP-regulated basolateral and apical Cl- transport pathways in T84 cells
Authors: Matthews J. B. , Awtrey C. S. , Hecht G. , Tally K. J. , Thompson R. S. , Madara J. L.
Published: December 2017

The effect of phorbol esters on adenosine 3',5'-cyclic monophosphate (cAMP)-regulated epithelial Cl- transport was studied in T84 cells, a human colonic cell line that serves as a model for electrogenic Cl- secretion. Preincubation of T84 cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a time- and dose-dependent inhibition of the net transepithelial secretory response to 10 microM forskolin (half-maximal inhibition at a concentration of approximately 10 nM PMA and a time of 45 min). Similar inhibition was observed with phorbol 12,13-dibutyrate but not the inactive phorbol ester phorbol 12,13-diacetate. Na(+)-K(+)-2Cl- cotransporter activity, assessed by bumetanide-sensitive 86Rb+ uptake, and K+ conductance, assessed by 86Rb+ efflux, were both found to be markedly reduced by PMA with a time course that paralleled the loss of the cAMP-regulated Cl- secretory response. One- and four-hour treatment of T84 cells with 100 nM PMA caused a sustained increase in the membrane-bound fraction of protein kinase C (PKC) but a decrease in total cellular PKC. Although, at these time points, the Na(+)-K(+)-2Cl- cotransporter and K+ efflux pathways were markedly inhibited (associated with inhibition of the forskolin-stimulated transepithelial Cl- secretory response), the activity of the cAMP-regulated Cl- efflux pathway, assessed by 125I-labeled efflux, remained unaffected. With prolonged exposure to PMA (up to 10), the cAMP-regulated Cl- efflux pathway was also eventually inhibited, and transepithelial electrical resistance progressively declined.(ABSTRACT TRUNCATED AT 250 WORDS)
Alpha-chymotrypsin deregulation of the sodium-calcium exchanger in barnacle muscle cells
Authors: Espinosa-Tanguma R. , DeSantiago J. , Rasgado-Flores H.
Published: December 2017

To gain insight into the mechanism by which the protease alpha-chymotrypsin (alpha-chym) activates the Na-Ca exchanger in muscle cells we studied 1) the ability of this enzyme to remove the intracellular "catalytic" Ca2+ requirement for activation of all the modes of exchange mediated by the Na-Ca exchanger (i.e., Nao-Cai, Nai-Cao, Nao-Nai, and Cao-Cai, where the subscripts o and i represent extracellular and intracellular, respectively), and 2) the ability of certain monovalent cations to stimulate Cao-Cai exchange after activation of the exchanger by alpha-chym. Barnacle muscle cells were used as models because these cells are so large that they can be internally perfused and voltage clamped. The results show that alpha-chym produces a highly activated Na-Ca exchanger able to operate in all its modes of exchange independently of catalytic Cai. The concentration-dependent effect of alpha-chym was biphasic; maximal activation occurred at 0.5 mg alpha-chym/ml perfusate for 20 min of perfusion at a perfusion rate of 2.5 microliters/min. The results are discussed in terms of the possible effects of alpha-chym on the kinetic modulation of the exchanger.
Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization
Authors: Geering K. , Jaunin P. , Jaisser F. , Merillat A. M. , Horisberger J. D. , Mathews P. M. , Lemas V. , Fambrough D. M. , Rossier B. C.
Published: December 2017

A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.
Amiloride-sensitive sodium conductance in human B lymphoid cells
Authors: Bubien J. K. , Warnock D. G.
Published: December 2017

The Na+ conductance of RPMI 8226 human B lymphoblastoid cells was examined using whole cell patch clamp. When the bath solution contained RPMI 1640 and the pipette solution contained (in mM) 100 potassium gluconate, 30 KCl, 10 NaCl, 0.5 ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), as well as < 10 nM free Ca2+, pH 7.25, the mean membrane potential was -58 +/- 4.5 mV (n = 18). Cells were voltage clamped from -160 to +40 mV in 20-mV increments. The inward conductance was 767 +/- 221 pS/10 pF, and the outward conductance was 1,212 +/- 272 pS/10 pF (n = 12). Superfusion with 2 microM amiloride significantly hyperpolarized the cells by 7.4 +/- 2.2 mV (P = 0.004), significantly reduced the inward conductance to 221 +/- 65 pS/10 pF (P = 0.028), but had no effect on the outward conductance (1,294 +/- 236 pS/10 pF, P = 0.820, after amiloride). Next, the pipette and bath solutions were changed to (in mM) 150 sodium gluconate, 0.5 EGTA, and 20 HEPES, as well as < 10 nM free Ca2+, pH 7.25. Under these conditions amiloride significantly reduced (50%, P < 0.05; n = 7) the whole cell currents. When potassium gluconate was substituted for sodium gluconate, amiloride had no effect. Thus amiloride inhibited a Na(+)-specific conductance expressed by B lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Emptying and refilling of Ca2+ store in tracheal myocytes as indicated by ACh-evoked currents and contraction
Authors: Janssen L. J. , Sims S. M.
Published: December 2017

Membrane currents and contractions evoked by acetylcholine (ACh) in freshly dissociated canine tracheal myocytes were investigated using the nystatin perforated-patch recording technique. In cells held at -60 mV in the presence of nifedipine, ACh evoked inward current (IACh) and contraction. Caffeine mimicked the effects of ACh. IACh and contractions could be evoked 3-4 min after removing external Ca2+ but were abolished by prolonged exposure to Ca(2+)-free media. Both responses were restored within minutes of reintroduction of Ca2+, even though the cells were held at -60 mV in the presence of nifedipine. IACh and ACh-evoked contractions were also reversibly abolished by continued exposure to caffeine. Cyclopiazonic acid (CPA), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced IACh by > 95% within 15 min but had little or no effect on the contractile responses evoked by ACh. IACh was restored after washout of CPA even though cells were held at -60 mV. After depleting the Ca2+ store with the use of CPA, depolarization of the membrane to +10 mV immediately before application of ACh led to a partial restoration of IACh. This restorative effect of depolarization was potentiated by Bay K 8644 and antagonized by nifedipine. In conclusion, IACh and contractions in canine tracheal myocytes are mediated by Ca2+ released from an internal store that can be depleted by prolonged removal of extracellular Ca2+, prolonged exposure to caffeine, or by blockade of the SR Ca(2+)-ATPase. At least two Ca2+ influx pathways appear to contribute to refilling of the internal store: one pathway that is not activated by depolarization or ACh and a second involving dihydropyridine-sensitive voltage-activated Ca2+ channels that may be in direct contact with the SR (i.e., conduct extracellular Ca2+ directly into the SR, bypassing the cytosol).
Metabolism of a long-chain diacylglycerol by permeabilized A10 smooth muscle cells
Authors: Chuang M. , Lee M. W. , Zhao D. , Severson D. L.
Published: December 2017

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG), was metabolized by cultured A10 smooth muscle cells after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue, dioctanoyl-glycerol ([3H]diC8), no appreciable 2-[14C]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[14C]POG was mainly converted into lipolytic products of a lipase pathway, monoacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via reactions catalyzed by either DAG acyltransferase, cholinephosphotransferase, or DAG kinase. Similar results were obtained in experiments with 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol. The conversion of 2-[14C]POG into PL and TG was not enhanced by the addition of 1 mM ATP-MgCl2, 1 mM CDP-choline, or 1 mM oleoyl-CoA to the alpha-toxin-treated A10 cells. The formation of FA and MG by permeabilized A10 cells was inhibited by DAG lipase inhibitors, U-57,908 (50 microM) and tetrahydrolipstatin (1-25 nM). The predominant contribution of the lipase pathway to the metabolism of a long-chain DAG, 2-[14C]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [3H]diC8 by intact A10 cells.
Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells
Authors: Chan H. C. , Fu W. O. , Chung Y. W. , Huang S. J. , Zhou T. S. , Wong P. Y.
Published: December 2017

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.
Effect of morphine on mesangial immunoglobulin G aggregate kinetics
Authors: Singhal P. C. , Pan C. Q. , Gibbons N. , Valderrama E.
Published: December 2017

Because mesangial expansion is considered a precursor of focal glomerulosclerosis, we studied whether morphine can cause mesangial expansion. We used radiolabeled human immunoglobulin G aggregates (125I-ahIgG) to study mesangial kinetics in control and experimental (morphine-treated) rats. Control and experimental rats were administered 125I-ahIgG by tail vein. Serum levels of 125I-ahIgG and uptake of 125I-ahIgG by liver, spleen, and mesangium were determined at 4, 8, 12, 24, and 36 h after 125I-ahIgG administration. Mesangial 125I-ahIgG levels were higher (P < 0.05) at 4 h and at later periods in morphine-treated vs. control rats. Naloxone, an opioid antagonist, did not attenuate the morphine-induced mesangial accumulation of 125I-ahIgG. The mean uptake of IgG aggregates was lower in the liver and spleen of morphine-treated rats at 36 h (P < 0.05). In both in vivo and in vitro experiments, ultrastructural studies showed accumulation of IgG-coated gold particles in vesicles, endosomes, and lysosomes. Morphine may have increased the accumulation of 125I-ahIgG in the glomeruli either by increasing the delivery of macromolecules into the mesangium or by altering the exit of macromolecules from the mesangium.
Possible specific activation of RNA synthesis in PC-12 cell isolated nuclei by small acidic peptides
Authors: Castigli E. , Mancinelli L. , Mariggio M. A. , Gianfranceschi G. L.
Published: December 2017

Three synthetic peptides, pyro-Glu-Ala-Gly-Glu-Ser-Glu-Asp (Pep A), pyro-Glu-Ala-Gly-Glu-Glu-Glu-Ser-Asn (Pep B), and pyro-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn (Pep C), bear sequences possibly belonging to components of a naturally occurring family of strongly related small acidic chromatin peptides involved in regulation of gene expression. In a crude nuclear fraction and in purified nuclei from PC-12 cells, Pep A and Pep B activate RNA synthesis, specifically acting on the RNA polymerase II transcription system. On the other hand, Pep C shows an inhibitory effect on RNA synthesis in purified nuclei but an activation in the crude nuclear fraction. Control experiments show that the serum thymic factor does not affect RNA synthesis in the crude nuclear fraction or in purified nuclei. A possible regulation by peptide phosphorylation via casein kinase II (more active in purified nuclei than in the crude nuclear fraction) is discussed.
pH heterogeneity during exercise in localized spectra from single human muscles
Authors: Vandenborne K. , Walter G. , Leigh J. S. , Goelman G.
Published: December 2017

We investigated whether pH heterogeneity in skeletal muscle during exercise, observed with 31P nuclear magnetic resonance, represents muscle fiber type heterogeneity. Localized spectra were simultaneously acquired from the soleus, medial, and lateral gastrocnemius using a multivolume localization technique, the Hadamard spectroscopic imaging (HSI) technique. Contamination of nonselected regions to the localized volumes was < 5%. HSI-localized spectra were obtained from the calf muscles of untrained subjects and a small group of athletes. Two plantar flexion exercise protocols were implemented: a "maximal" high frequency protocol and a "steady-state" protocol at low contraction frequency (0.25 Hz). pH heterogeneity was observed in localized spectra of single muscles during both exercise protocols, as indicated by the large Pi line width. During maximal exercise the Pi line width was up to three times wider than the phosphocreatine line width, covering an entire pH unit. During the steady-state exercise, in three subjects the Pi peak clearly resolved into two distinct peaks, one at low pH and one at high pH. As pH heterogeneity was observed in localized spectra of single muscles during both exercise protocols, it most likely reflects the metabolic heterogeneity between fiber types.
Nanomolar ouabain augments caffeine-evoked contractions in rat arteries
Authors: Weiss D. N. , Podberesky D. J. , Heidrich J. , Blaustein M. P.
Published: December 2017

Chronic parenteral administration of ouabain to normal rats raises plasma ouabain concentrations to low nanomolar levels and induces hypertension [C. M. Yuan, P. Manunta, J. M. Hamlyn, S. W. Chen, E. Bohen, J. Yeun, F. J. Haddy, and M. B. Pamnani. Hypertension 22: 178-187, 1993 and see also M. P. Blaustein. Am. J. Physiol. 264 (Cell Physiol. 33): C1367-C1387, 1993]. To determine whether rat arteries are sensitive to these low ouabain levels, we tested the effects of various ouabain concentrations on caffeine-evoked contractions (CEC) in rat aortic and small mesenteric artery rings. CEC amplitude was used as a measure of the sarcoplasmic reticulum (SR) Ca2+ content. Ouabain increased CEC in aortic as well as mesenteric artery rings, but the effects in the aorta were difficult to quantitate because the CEC were often oscillatory. Mesenteric artery, under control conditions and after sensitization with 10-30 nM phenylephrine (PE), exhibited biphasic ouabain dose-CEC response curves. Low concentrations of ouabain (0.1-10 nM) caused small significant increases in CEC, but a further effect was observed only with > or = 10 microM ouabain. PE shifted the ouabain dose-response curve toward lower ouabain concentrations; conversely, ouabain shifted the PE dose-response curve toward lower PE concentrations. It appears that nanomolar concentrations of ouabain can influence vascular responsiveness to vasoconstrictors. We conclude that rat vascular smooth muscle contains both high- and low-affinity ouabain receptors, possibly corresponding to Na+ pumps with alpha 3- and alpha 1-subunit isoforms, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)