bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased aerobic glucose oxidation by cAMP in cultured regenerated skeletal myotubes
Authors: Freerksen D. L. , Schroedl N. A. , Johnson G. V. , Hartzell C. R.
Published: December 2017

Previous studies of embryonic rat skeletal muscle cultures suggested that there was a correlation between intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration and activities of enzymes of oxidative energy metabolism. We investigated the ability of agents that elevate intracellular cAMP by three different mechanisms (activation of adenylate cyclase, inhibition of phosphodiesterase, and analogues) to alter not only the activities of 11 glycolytic and mitochondrial enzymes but also the rate of flux through aerobic glucose oxidation in intact myotubes derived from regenerating rat muscle satellite cells. The enzyme activities were not consistently altered when cAMP was elevated, with the exception of the electron transport enzyme, NADH cytochrome c reductase, whose activity was elevated by exposure of the myotubes to cholera toxin (110% of control), 3-isobutyl-1-methylxanthine (112%), caffeine (119%), and 8-bromoadenosine 3',5'-cyclic monophosphate (120%). The rate of flux of aerobic glucose oxidation was elevated by all agents (116-157% of control) except cholera toxin. This study allowed us to compare the metabolic characteristics of myotube cultures derived from satellite cells with those from embryonic muscle, from a previous study. Despite differences between these two models, together, the data present strong evidence that an increase in intracellular cAMP can cause an increase in oxidative capacity.
Energy metabolism of skeletal muscle biopsies stimulated anaerobically without load in vitro
Authors: Young D. A. , Chi M. M. , Lowry O. H.
Published: December 2017

This study was made to test the validity of a simple biopsy technique for assessing the metabolic capacity of skeletal muscle. The biopsy is stimulated under mineral oil without attachment, i.e., without load or tension, then freeze-clamped and assayed for ATP, phosphocreatine, glucose 6-phosphate, and lactate. The mineral oil creates a closed anaerobic system. Background studies demonstrated in the absence of a load, metabolic changes with stimulation were little affected by cutting the fibers to obtain the biopsy; and high-energy phosphate (approximately P) consumption during a brief tetanus was not much lower than that for an isometric tetanus. Individual fast-twitch oxidative-glycolytic (IIA) and fast-twitch glycolytic (IIB) fibers obtained from the freeze-clamped biopsy showed distinct differences in approximately P consumption and metabolic changes. The results indicate that this technique could be useful for studies of normal and pathological human muscle.
Effects of glucocorticoid treatment on cardiac protein synthesis and degradation
Authors: Clark A. F. , DeMartino G. N. , Wildenthal K.
Published: December 2017

We treated rats with dexamethasone (DEX, 1 mg . kg-1 . day-1) and examined the effects of this glucocorticoid on heart protein metabolism using atrial explant and Langendorff perfusion preparations. Fasted rats treated with DEX for 2 days had significantly lower body weights (92% of control, P less than 0.001) and larger hearts (106% of control, P less than 0.005) than fasted control animals. Protein and RNA concentrations remained constant. In atrial explants, DEX treatment produced a 19% increase in protein synthesis (P less than 0.001) and a 13% increase in protein degradation (P less than 0.002). In Langendorff-perfused hearts, DEX treatment caused a 36% increase in protein synthesis (P less than 0.02), while protein degradation was 8% above control (P greater than 0.05). Thus, in contrast to their catabolic effects on skeletal muscle, glucocorticoids are anabolic on the heart. The increased accumulation of total cardiac protein during early glucocorticoid administration is mediated entirely via increased rates of synthesis.
Water permeability of alveolar macrophages
Authors: Garrick R. A. , Polefka T. G. , Cua W. O. , Chinard F. P.
Published: December 2017

The hydraulic conductivity coefficient (Lp) of alveolar macrophages, recovered by lavage from dog lungs, was determined by following volume changes induced by changes of nonpermeating solute concentrations of suspending fluid as a function of time at 20 degrees C. The volume changes were monitored as changes in absorbance of the suspended cells at 600 nm. Cell surface area was calculated from cell volume and diameter. Linear relationships between cell volume and solution osmolality changes were found over the range of 320-520 mosmol/kg; beyond these ranges the macrophages did not respond with swelling or shrinking. Lp and the filtration coefficient (Pf) were calculated from the total volume change over time. At 20 degrees C these were, respectively, 15.7 X 10(-10) cm X cmH2O-1 X s-1 and 217 X 10(-5) cm/s. Comparison of Pf and the diffusional permeability coefficient (Pd) for water of 70 X 10(-5) cm/s, yields a Pf-to-Pd ratio of 3.1. The hypothesis of water passage through aqueous membrane pores is compatible with these data. However, diffusion of water in the glycocalyx of the pericellular domain could be restricted. Pd would then be underestimated, and a falsely high ratio would be calculated. We have no evidence to support this possibility.
Target molecular weights for red cell band 3 stilbene and mercurial binding sites
Authors: Verkman A. S. , Skorecki K. L. , Jung C. Y. , Ausiello D. A.
Published: December 2017

Radiation inactivation was used to measure the target sizes for binding of disulfonic stilbene anion transport inhibitor 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) and mercurial water transport inhibitor p-chloromercuribenzene sulfonate (pCMBS) to human erythrocytes. The measured target size for erythrocyte ghost acetylcholinesterase was 78 +/- 3 kDa. DBDS binding to ghost membranes was measured by a fluorescence enhancement technique. Radiation (0-26 Mrad) had no effect on total membrane protein and DBDS binding affinity, whereas DBDS binding stoichiometry decreased exponentially with radiation dose, giving a target size of 59 +/- 4 kDa. H2-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2-DIDS, 5 microM) blocked greater than 95% of DBDS binding at all radiation doses. pCMBS binding was measured from the time course of tryptophan fluorescence quenching in ghosts treated with the sulfhydryl reagent N-ethylmaleimide (NEM). Radiation did not affect the kinetics of tryptophan quenching, whereas the total amplitude of the fluorescence signal inactivated with radiation with a target size of 31 +/- 6 kDa. These results support the notion that DBDS and pCMBS bind to the transmembrane domain of erythrocyte band 3 in NEM-treated ghosts and demonstrate that radiation inactivation may probe a target significantly smaller than a covalently linked protein subunit. The small target size for the band 3 stilbene binding site may correspond to the intramembrane domain of the band 3 monomer (52 kDa), which is physically distinct from the cytoplasmic domain (42 kDa).
Kinetics of the Na+-H+ antiporter as assessed by the change in intracellular pH in MDCK cells
Authors: Selvaggio A. M. , Schwartz J. H. , Bengele H. H. , Alexander E. A.
Published: December 2017

The Na+-H+ antiporter regulates both H+ secretion and cell pH in renal epithelia. The present study was designed to further define the Na+ dependency and kinetics of proton efflux in MDCK cells. Intracellular pH (pHi) was determined with a pH-sensitive fluorescent probe, 2,7-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF). Data were obtained from confluent monolayers grown on plastic cover slips and studied in media free of added CO2 and HCO-3, pH = 7.2 (pHo). Monolayers in NaCl maintain a pHi of 7.48 +/- 0.03 (n = 13). When cells were acidified with NH4Cl, pHi decreased to 6.73 +/- 0.07 (n = 10) and remained stable in Na+-free choline chloride. When monolayers were subsequently exposed to NaCl, pHi increased 0.28 +/- 0.04 pH units/min. Amiloride (2.5 mM) inhibited this Na+-dependent rise in pHi by more than 75%. The rate of pHi recovery after exposure to Na+ exhibited saturation kinetics; the apparent Km(Na) was 30.4 +/- 4.2 mM and maximum velocity was 107.1 +/- 17.1 delta [H+]i nmol X l-1 X min-1. We conclude that pHi regulation in the MDCK cell is in part mediated by a Na+-H+ antiporter, and the kinetics of this process can be reliably assessed by the pH-sensitive fluorometric probe, BCECF.
Adenosine 3',5'-cyclic monophosphate stimulates chloride secretion in A6 epithelia
Authors: Yanase M. , Handler J. S.
Published: December 2017

Basal and aldosterone-stimulated short-circuit current (Isc) of A6 epithelia are known to be equivalent to net apical to basal Na flux and are completely inhibited by 0.05 mM amiloride added to the solution bathing the apical surface of the epithelium. In the absence of amiloride, the Isc stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) is also equivalent to net apical to basal Na flux. However, amiloride does not completely inhibit the cAMP-stimulated Isc. In this study, the cAMP-stimulated, amiloride-insensitive Isc was characterized, using vasopressin or forskolin to raise cell cAMP. After basal Isc is inhibited by amiloride, forskolin stimulates Isc, conductance, and bidirectional 36Cl flux. Stimulation of Isc depends on the presence of both Na and Cl; stimulation of conductance depends on the presence of Cl. 36Cl flux studies showed that the cAMP-stimulated, amiloride-insensitive Isc is equivalent to net Cl flux. It is inhibited by ouabain and by furosemide or bumetanide added to the solution bathing the basal surface of the epithelium. In view of the effect of cAMP in some other epithelia, we suggest that cAMP activates apical membrane Cl channels that are in series with a Na-K-Cl cotransporter in the basolateral plasma membrane.
Na+-H+ exchanger of human placental brush-border membrane: identification and characterization
Authors: Balkovetz D. F. , Leibach F. H. , Mahesh V. B. , Devoe L. D. , Cragoe E. J. , Ganapathy V.
Published: December 2017

Syncytiotrophoblast brush-border membrane vesicles isolated from full-term human placentas were shown to transport Na+ against a concentration gradient in the presence of an outward proton gradient [( H+i] greater than [H+]o). This proton gradient-coupled Na+ uptake was markedly inhibited and the uphill transport abolished when the electrochemical proton gradient was dissipated by carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone. The presence of nigericin also eliminated the concentrative uptake of Na+ in these vesicles. Dimethylamiloride and harmaline inhibited the proton gradient-induced Na+ uptake. The apparent inhibition constant for this process was 0.32 microM for dimethylamiloride was freely reversible and the inhibitor reduced the Na+ uptake by directly interacting with the exchanger protein rather than by dissipating the H+ gradient. The dimethylamiloride-sensitive Na+ uptake was saturable with respect to Na+. The affinity constant for Na+ was 7.8 +/- 1.2 mM and the maximal velocity was 38.7 +/- 2.4 nmol X mg protein-1 X min-1. The dimethylamiloride-insensitive Na+ uptake was not saturable and probably represented simple diffusion. The diffusional component accounted for only 10% of the total uptake. Li+ strongly competed with Na+ for the uptake process and the apparent inhibition constant was 3.6 +/- 0.4 mM. Tetraethylammonium also caused significant inhibition of Na+ uptake, whereas K+, Rb+, Cs+, and choline had no effect. These data provide evidence for the existence of a Na+-H+ exchanger in human placental brush-border membrane, and the properties of this exchanger are similar to those of the Na+-H+ exchanger identified in the brush-border membrane of mammalian kidney and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)
Physiological changes induced in cardiac myocytes by cytotoxic T lymphocytes
Authors: Hassin D. , Fixler R. , Shimoni Y. , Rubinstein E. , Raz S. , Gotsman M. S. , Hasin Y.
Published: December 2017

The “lethal hit” induced by viral specific, sensitized, cytotoxic T lymphocytes (CTL) attacking virus-infected heart cells is important in the pathogenesis of viral myocarditis and reflects the key role of CTL in this immune response. The mechanisms involved are incompletely understood. Studies of the physiological changes induced in mengovirus-infected, cultured, neonatal, rat heart cells by CTL that had been previously sensitized by the same virus are presented. The CTL were obtained from spleens of mengovirus-infected, major histocompatibility complex (MHC) matched adult rats. Cell wall motion was measured by an optical method, action potentials with intracellular microelectrodes, and total exchangeable calcium content by 45Ca tracer measurements after loading the myocytes with 45Ca and then exposing them to CTL. After 50 min (mean time) of exposing mengovirus-infected myocytes to the CTL, the mechanical relaxation of the myocyte was slowed, with a subsequent slowing of beating rate and a reduced amplitude of contraction. Impaired relaxation progressed, and prolonged oscillatory contractions lasting up to several seconds appeared, with accompanying oscillations in the prolonged plateau phase of the action potentials. Arrest of the myocyte contractions appeared 98 min (mean time) after exposure to CTL. These changes in action potentials and contractions were reversible either by washout with the normal medium or by the addition of verapamil. The amount of total exchangeable calcium in the cultured myocytes, 1 h after exposure to CTL, was significantly increased. This increase was prevented by pretreatment with verapamil. (ABSTRACT TRUNCATED AT 250 WORDS)
Modulation of Na+-Ca2+ exchange in sarcolemmal vesicles by intravesicular Ca2+
Authors: Reeves J. P. , Poronnik P.
Published: December 2017

When cardiac sarcolemmal vesicles were incubated at 37 degrees C in 160 mM NaCl containing 0.5 mM CaCl2 and subsequently assayed for Na-Ca exchange activity, they exhibited a threefold increase in the initial rate of 45Ca2+ uptake (at 20 microM 45Ca2+) compared with vesicles incubated without added CaCl2. Removal of endogenous Ca2+ by incubation of the vesicles with 0.1 mM ethylene-bis(beta-aminoethylether)N,N'-tetraacetic acid (EGTA) resulted in a 35% inhibition in exchange activity. The pretreatment with CaCl2 produced an acceleration of Na-Ca exchange activity rather than an increase in Ca2+ uptake due to Ca-Ca exchange. Pretreatment of the vesicles with CaCl2 lowered the apparent Km of the exchange system for Ca2+. The effects of the Ca treatment were reversed by subsequently incubating the vesicles with EGTA. In contrast to the effects of intravesicular Ca2+ on Nai-dependent Ca2+ uptake, external Ca2+ had no effect on Nao-dependent Ca2+ efflux. The results suggest that an understanding of the kinetics of the Na-Ca exchange system may be hampered by the autoacceleration of exchange activity that occurs during initial rate measurements as Ca2+ accumulates within the vesicles. This phenomenon may contribute to the variability that exists among different vesicle preparations in their apparent Km values for Ca2+.
High cytosolic pH inhibits Ca uptake by Myxicola axon mitochondria
Authors: Abercrombie R. F. , Gammeltoft K.
Published: December 2017

Microliter samples of cytoplasm containing mitochondria were aspirated from giant axons of the marine annelid Myxicola infundibulum into polyethylene tubes. The small molecular constituents within these cytoplasmic samples were controlled by a dialysis capillary with a 6,000 molecular weight cut off. The negative log of the calcium ion activity (pCa) (6.72 +/- 0.03, n = 40) and, in some cases, the pH (7.51 +/- 0.01, n = 7) of the samples were monitored with ion-sensitive microelectrodes. Adding 5 mM succinate or 5 mM ATP at pH 7.5 caused the Ca activity in the cytoplasm to drop from an experimentally elevated value of approximately 10 microM to below 1 microM. This decrease could be inhibited with ruthenium red, suggesting a mitochondrial mechanism. Ca uptake, following the addition of either succinate or ATP, was reversibly slowed when the cytoplasmic pH was elevated to approximately 8.3. When ruthenium red was added after mitochondria had taken up Ca, the Ca activity in the extramitochondrial cytoplasm gradually increased suggesting ongoing release of Ca from storage sites. Increasing the cytoplasmic pH to approximately 8.5 in the presence of ruthenium red did not increase the ongoing release over that found with ruthenium red alone. The apparent washout of Ca from the energy-independent, nonmitochondrial Ca buffers was only slightly affected by pH (pH 7.5-8.5). It is concluded that elevating intracellular pH to 8.3 slows the Ca uptake by mitochondria. Thus cytoplasmic pH may have a function in regulating mitochondrial Ca metabolism and/or extramitochondrial calcium activity.
Sodium permeability of frog skeletal muscle in absence and presence of veratridine
Authors: McKinney L. C. , Ratzlaff R. W.
Published: December 2017

The effect of veratridine on the Na permeability of frog sartorius muscle was studied by means of ion flux measurements using radiolabeled sodium. Veratridine increases Na influx in a dose-dependent manner (apparent Kd = 160 +/- 7 microM when Vm congruent to -40 mV). The increase can be completely inhibited by tetrodotoxin (TTX) (apparent Ki = 8 +/- 2 nM), indicating that all veratridine-induced Na influx occurs via sodium channels. The time constant for the rate of onset of veratridine action is 1 h. Raising external pH one unit to 8.3 causes the rate of action of veratridine and the final level of Na influx to increase. The apparent Kd for veratridine depends on membrane voltage. Values obtained in 2.5 and 5 mM K Ringer (Vm congruent to -95 and -80 mV) were 579 +/- 279 and 35 +/- 8 microM, respectively. Veratridine-induced Na influx obeys the Goldman constant field flux equation and when veratridine concentration is 1 mM, sodium permeability is 1.5 X 10(-7) cm/s. This is much less than the maximum PNa (1.6 X 10(-3) cm/s) obtained from voltage-clamp measurements of peak Na conductance. Mg (52 mM) inhibits veratridine-induced influx by about half. Aspects of resting Na influx in the absence of veratridine (but in the presence of ouabain) were also characterized. Steady-state Na influx is unaffected by tetrodotoxin over the voltage range -90-0 mV, suggesting that no sodium channels are open in the resting state. Na influx is also insensitive to curare. It is linearly dependent on external sodium and larger at more negative membrane potentials.
Interaction of two electrical pacemakers in muscularis of canine proximal colon
Authors: Smith T. K. , Reed J. B. , Sanders K. M.
Published: December 2017

Experiments were performed to determine the source of the 20 cycles/min electrical oscillation commonly seen in colonic electrical records, the influence of the 20 cycles/min rhythm on the circular and longitudinal muscle layers, and the interactions between the 20 cycles/min rhythm and slow waves in circular muscle cells. Cross-sectional muscle preparations of the canine proximal colon were used to allow impalement of cells at any point through the thickness of the muscularis. Intracellular recordings from circular muscle cells clearly showed the two characteristic pacemaker frequencies in the colon (6 cycles/min slow waves; 20 cycles/min oscillations). The 20 cycles/min oscillations were recorded from longitudinal and circular muscle cells. Their amplitudes were greatest at the myenteric border. In the longitudinal layer the 20 cycles/min events initiated action potentials; in circular muscle the 20 cycles/min events summed with slow waves. Simultaneous recordings from circular and longitudinal cells across the myenteric border demonstrated that events in the two layers were usually in phase, suggesting that the two layers are electrically coupled and are paced by a common pacemaker. The amplitude of the 20 cycles/min events decayed with distance from the myenteric border in both circular and longitudinal muscles. The data demonstrate that two discrete populations of pacemaker cells generate the spontaneous electrical activity in the colon. Both events appear to passively spread through the circular muscle. It is the summation of these events that appears to serve as the signal for excitation-contraction coupling in circular muscle.
Cell volume regulation in hemoglobin CC and AA erythrocytes
Authors: Berkowitz L. R. , Orringer E. P.
Published: December 2017

Swelling hemoglobin CC erythrocytes stimulates a ouabain-insensitive K flux that restores original cell volume. This volume regulatory pathway was characterized for its anion dependence, sensitivity to loop diuretics, and requirement for Na. The swelling-induced K flux was eliminated if intracellular chloride was replaced by nitrate and both swelling-activated K influx and efflux were partially inhibited by 1 mM furosemide or bumetanide. K influx in swollen hemoglobin CC cells was not diminished when Na in the incubation medium was replaced with choline, indicating Na independence of the swelling-induced flux. Identical experiments with hemoglobin AA cells also demonstrated a swelling-induced increase in K flux, but the magnitude and duration of this increase were considerably less than that seen with hemoglobin CC cells. The increased K flux in hemoglobin AA cells was likewise sensitive to anion replacement and to loop diuretics and did not require the presence of Na. These data indicate that a volume-activated K pathway with similar transport characteristics exists in both hemoglobin CC and AA red cells.
Mutant isolation and gene transfer as tools in study of transport proteins
Authors: Gargus J. J.
Published: December 2017

Somatic cell and molecular genetic concepts, techniques, and approaches relevant to the analysis of a complex mechanism such as a mammalian ion transport system are reviewed and illustrated with examples taken from studies of K+ transport mutants. The reasons for undertaking a study of mutants and mutations are stated and followed by directly applicable approaches to transport mutant isolation. Examples of the types of information that can be extracted from a characterization of such mutants is then presented. The parasexual system of somatic cell hybridization is reviewed, again, followed by applicable approaches and examples of how data from such studies can be used. Finally, the technique of DNA-mediated gene transfer is presented as a method that allows the actual isolation of a transporter gene which has been characterized as above.
Selectable mutations altering two mechanisms of mammalian K+ transport are dominant
Authors: Gargus J. J.
Published: December 2017

Somatic cell mutants with altered K+ transport have previously been isolated from mutagenzied LMTK- cells for their ability to grow at subthreshold low-potassium concentrations (0.2 mM). These mutants fall into two classes: one class, LTK-5, possesses a functionally altered furosemide-sensitive Na+-K+-Cl- cotransport system and the other, LTK-1, an altered K+-conducting channel. Somatic cell hybrids have been formed between each of these cell lines and a wild-type L-cell line, making use of complementing selectable marker mutations carried by these parents, to establish the dominance of the K+ transport mutations. Hybrids were isolated and studied in two ways: clonal hybrid cell lines were selected in a manner unbiased toward their K+ transport phenotype, which was later assayed; and the number of independent hybrids arising in this single-selective condition was compared with the number arising in a condition which is double selective for the mutant phenotype as well. By both assays, hybrids formed with LTK-1 or LTK-5 as a parent uniformly exhibited the mutant phenotype by growth and cloning, whereas control hybrids with LMTK- as parent never did. This demonstrates both transport mutations to be dominant and thus potentially isolatable.
Effects of Ca2+, Mg2+, and myosin phosphorylation on skinned smooth muscle fibers
Authors: Barsotti R. J. , Ikebe M. , Hartshorne D. J.
Published: December 2017

Isometric tension, unloaded shortening velocity (Vus), and myosin light chain phosphorylation were measured with skinned chicken gizzard fibers at various Ca2+ concentrations and at two concentrations of free Mg2+, 0.7 and 2.2 mM. At low free Mg2+, an increase in Ca2+ from pCa 8.0 to 6.4 resulted in an increase of all three parameters. Between pCa 6.4 and 5.0, isometric tension and phosphorylation remained constant but Vus continued to increase. At low free Mg2+, therefore, Vus showed a dependence both on phosphorylation and on Ca2+. At high free Mg2+, tension and Vus increased as phosphorylation increased and both were maximum at pCa 6.4, where phosphorylation became constant. Therefore, at high free Mg2+, Vus was dependent only on phosphorylation and did not show an additional Ca2+ dependence. Incubation of the Ca2+-independent kinase (approximately 3 microM) with skinned fibers under various conditions resulted in a constant level of phosphorylation (49-58%). At high free Mg2+ plus the Ca2+-independent kinase Vus was independent of Ca2+, whereas at low free Mg2+ Vus increased from pCa 6.4 to 5.0. These data are consistent with the hypothesis that Ca2+ binding to the Ca2+-Mg2+ sites of myosin increase Vus and that this occurs at Ca2+ concentrations higher than those necessary to saturate calmodulin.
Pituitary Ca2+ channels: blockade by conventional and novel Ca2+ antagonists
Authors: Enyeart J. J. , Sheu S. S. , Hinkle P. M.
Published: December 2017

We have identified several new agents that block Ca2+ channels in the rat pituitary GH4C1 cell line. These drugs, which include the diphenylbutylpiperidine antipsychotic pimozide, the calmodulin antagonist calmidazolium, and the steroidal Na+ channel toxin veratridine, were compared with several conventional Ca2+ antagonist in 45Ca2+ uptake, prolactin secretion, and whole cell patch voltage-clamp experiments. Pimozide, the most potent of these novel Ca2+ antagonists, inhibited depolarization-dependent 45Ca2+ uptake and prolactin secretion half maximally at a concentration of 100 nM, whereas calmidazolium and veratridine produced 50% inhibition at concentrations of 500 nM and 1 microM. In comparison, the three organic Ca2+ antagonists nitrendipine, verapamil, and diltiazem blocked 45Ca2+ uptake half maximally at concentrations of 2.5 nM, 1 microM, and 2.5 microM, respectively. All of the antagonists inhibited Ca2+ uptake and prolactin secretion stimulated by the dihydropyridine Ca2+ agonist BAY-K 8644 less potently than KCl-stimulated responses. In patch-clamp experiments, pimozide, veratridine, and nitrendipine blocked Ca2+ current through the slowly inactivating Ca2+ channels of GH4C1 cells. These results demonstrate that Ca2+ channels in an endocrine cell line can be blocked by a variety of molecules including sodium channel toxins and calmodulin antagonists. The data extend the pharmacological similarity between Ca2+ channels in pituitary and other excitable cells and suggest a structural similarity among several cellular proteins.
Na-H and Cl-HCO3 exchange in rabbit oxyntic cells using fluorescence microscopy
Authors: Paradiso A. M. , Tsien R. Y. , Demarest J. R. , Machen T. E.
Published: December 2017

We have used the fluorescence intensity ratio (excitation 490/439; emission 520-550 nm) of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to measure intracellular pH (pHi) in single oxyntic cells (OCs) within intact gastric glands isolated from rabbits. The fluorescence ratio was converted to pHi by exposing cells to high [K] Ringer plus 10(-5) M nigericin. Ratios varied linearly with pHi over the physiological range. When the bathing solution was changed from NaCl to Na gluconate Ringer, pHi increased from 7.0 to 7.4. The increase of pHi occurred equally rapidly in nominally CO2-HCO3-free solutions and in solutions containing 5% CO2 and 25 mM HCO3. This effect was reversible and blocked by 2 X 10(-4) M 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). The DIDS block was bypassed by 10(-5) M tri-n-butyltin, a pharmacological Cl-OH exchanger. When the bathing solution was changed from NaCl to N-methyl-D-glucamine (NMG) Cl Ringer, pHi decreased reversibly from 7.0 to 6.8. It was also found that changing from NMG gluconate to Na gluconate Ringer caused pHi to increase from 7.1 to 7.3, and this alkalinization was blocked by 10(-3) M amiloride; changing from NMG gluconate to NMG Cl Ringer caused pHi to decrease to 6.7. We conclude that OCs contain separate Na-H and Cl-HCO3 exchangers. Similarities and differences between these exchangers and those present in other cell types are discussed.
Interactions of sodium-proton exchange mechanism in dog red blood cells with N-phenylmaleimide
Authors: Parker J. C. , Glosson P. S.
Published: December 2017

Dog red blood cells (RBC) have a Na-H exchanger that is reversibly activated by cell shrinkage. The Na-H exchanger can be fixed in the on or off mode by treating the cells with N-phenylmaleimide. This action depends on the volume of the cells at the time of exposure to N-phenylmaleimide and also on the concentration of the reagent per number of cells. If the cells are swollen in hypotonic media during N-phenylmaleimide exposure, the Na-H exchanger becomes irreversibly inactivated, so that on subsequent shrinkage of the cells, no amiloride-sensitive Na flux is seen. This effect is maximal at N-phenylmaleimide concentrations of greater than 20 mumol/g hemoglobin. If the cells are shrunken in hypertonic media during N-phenylmaleimide exposure, the response of the Na-H exchanger depends critically on the concentration of the reagent. At N-phenylmaleimide concentrations of less than 20 mumol/g hemoglobin, the Na-H exchanger is fixed in the activated state, so that even when the volume stimulus is removed by subsequent cell swelling, an amiloride-sensitive flux is seen. Higher concentrations of N-phenylmaleimide applied to shrunken cells inhibit the Na-H exchanger. The results are accounted for in a model that envisions a volume-responsive switching mechanism for Na-H exchange that has two functional groups capable of reacting with N-phenylmaleimide. The accessibility of these groups is determined by cell volume.
Na-H antiport in cultured rat aortic smooth muscle: its role in cytoplasmic pH regulation
Authors: Weissberg P. L. , Little P. J. , Cragoe E. J. , Bobik A.
Published: December 2017

We have investigated the role of the Na-H antiport in the regulation of intracellular pH (pHi) in vascular smooth muscle. Experiments were conducted on contractile-state rat aortic smooth muscle cells grown in primary culture and loaded with the pH-sensitive, fluorescent indicator 2',7',-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Cells equilibrated in a normal physiological salt solution (PSS) containing 135 mM Na, pH 7.4 at 37 degrees C, had a pHi of 7.16 +/- 0.04 (means +/- SE; n = 8). 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a concentration-dependent fall in pHi. Removal of extracellular Na caused an intracellular acidification that was rapidly reversed on replacement of Na. The rate of recovery from NH4Cl-induced intracellular acidosis was dependent on extracellular Na concentration (Km 14.6 +/- 2.8 mM) and was accelerated by increasing the transmembrane Na gradient and slowed by decreasing it. Recovery from acidosis was completely abolished by either EIPA or the absence of extracellular Na. These results demonstrate that the Na-H antiport is an important mechanism for the maintenance and regulation of pHi in vascular smooth muscle cells. The BCECF fluorescence technique provides an ideal method for further studies on the mechanisms for pHi regulation in these cells.
High NaCl induces stable changes in phenotype and karyotype of renal cells in culture
Authors: Uchida S. , Green N. , Coon H. , Triche T. , Mims S. , Burg M.
Published: December 2017

Extracellular fluid in the renal medulla normally is hyperosmotic. To test adaptation to such an environment, a continuous line of rabbit renal inner medullary epithelial cells (GRB-PAP1), which had been established in isosmotic medium, was switched to a medium containing high NaCl. The origin of these cells is described. When the osmolality was raised from 300 to 600 mosmol/kg by adding NaCl, cells eventually survived and proliferated, but unexpectedly, they underwent major changes in phenotype and karyotype that persisted during proliferation in isosmotic or hyperosmotic medium for at least 7 months. The threshold concentration for the changes was approximately 500 mosmol/kg. Cells of a typical strain (PAP-HT25) that formed in hyperosmotic medium were much larger and more often multinucleated than were GRB-PAP1. GRB-PAP1 cells were near diploid; PAP-HT25 cells were polyploid. The changes, since they occurred in most clones, were due to adaptation of the majority of cells and not to selection of a minority of cells already having these characteristics. Cloning efficiency was higher for GRB-PAP1 than PAP-HT25 in isosmotic medium, but the reverse occurred in hyperosmotic medium. Thus exposure to the hyperosmotic medium induced greater ability to clone in it. We suggest that these changes may involve persistent alterations in gene regulation, possibly like those previously reported in chicken embryo fibroblast cells after hyperosmotic NaCl (Cell 30: 131-139, 1982).
Beta-adrenergic relaxation of smooth muscle: differences between cells and tissues
Authors: Scheid C. R.
Published: December 2017

The present studies were carried out in an attempt to resolve the controversy about the Na+ dependence of beta-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of beta-adrenergic agents, including a stimulatory effect on 45Ca efflux, were dependent on the presence of a normal transmembrane Na+ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of beta-adrenergic agents was Na+ independent. Uncertainty remained as to whether these discrepancies reflected differences between cells and tissues or differences between species. Thus, in the present studies, we utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na+ dependence of beta-adrenergic relaxation. We found that elimination of a normal Na+ gradient abolished beta-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Rather they may be attributable to unique properties of tissues, such as the presence of pacemaker cells, nerves, and so on. Thus the controversy concerning the mechanism of beta-adrenergic relaxation may reflect inherent differences between tissues and cells.
Dependence on multivalent cations of quantal release of transmitter induced by black widow spider venom
Authors: Misler S. , Falke L. C.
Published: December 2017

Application of alpha-latrotoxin (alpha-LT), the active component of black widow spider venom (BWSV), to a vertebrate neuromuscular junction, in the presence of millimolar bath concentrations of Ca2+ or Mg2+, greatly increases the frequency of miniature end-plate potentials (Fmepp). We have further characterized the cation dependence of alpha-LT action at the frog cutaneous pectoris neuromuscular junction. The divalent cations, Ca, Sr, Ba at less than or equal to 50 microM, Zn, Mn, Cd at greater than or equal to 50-100 microM, and Mg at greater than or equal to 1.0 mM, as well as the trivalent cation La at greater than or equal to 15 microM, all increase Fmepp exponentially to greater than or equal to 100-200 s-1 over several minutes time. The exponential rate of rise is graded with extracellular cation concentration and can be reduced by increasing [K+] of the bath from 2 to 25-40 mM. Long-term exposure to alpha-LT in the presence of Sr2+ or Mn2+ results in the exhaustion of the releasable quantal store of transmitter, which in the case of Mn2+ correlates well with depletion of synaptic vesicles. These data support the hypothesis that BWSV promotes an increase in Fmepp by increasing nerve terminal permeability to multivalent cations that enter the nerve terminal down their electrochemical gradients and then may bind to quantal release activating sites or displace Ca2+ from intracellular stores.
Structural and functional relationship of red blood cell protein 4.1 to synapsin I
Authors: Krebs K. E. , Prouty S. M. , Zagon I. S. , Goodman S. R.
Published: December 2017

It has been suggested that the neuron specific protein synapsin I is closely related to red blood cell (rbc) protein 4.1. A systematic comparison of the structural and functional properties of rbc protein 4.1 and synapsin I has been carried out. There is approximately a three order of magnitude difference in cross reactivity of synapsin I with rbc 4.1 antiserum vs. synapsin I antiserum, as determined by a competitive quantitative dot assay. Two-dimensional chymotryptic iodopeptide mapping analysis demonstrated limited peptide homology (approximately 34% spot overlap) between rbc 4.1 and synapsin I. Dephosphorylated synapsin I binds saturably to brain spectrin (240/235) with an estimated dissociation constant (Kd) of 700 nM and a maximal binding capacity of 4 mol synapsin I/mol spectrin tetramer, similar to the affinity and stoichiometry of 4.1 binding to rbc spectrin. Synapsin I was found to bind to the terminal ends of the brain spectrin tetramer by low-angle rotary shadowing, analogous to 4.1 binding to rbc spectrin. In summary, synapsin I is structurally and immunologically distinct from rbc 4.1, yet shares functional similarities with rbc 4.1 with respect to its spectrin binding characteristics.
Renal epithelial cell growth can occur in absence of Na+-H+ exchanger activity
Authors: Mohrmann M. , Cantiello H. F. , Ausiello D. A.
Published: December 2017

An electroneutral Na+-H+ exchange system has been described in a variety of tissues and cell types, including those of renal origin, and has been proposed to play a role in the activation of growth. We have recently characterized the presence of this ubiquitous transporter in the apical domain of confluent epithelial LLC-PK1 cells (J. Biol. Chem. 261: 3252-3258, 1986). Because most apical membrane proteins appear late in cell growth, accompanying epithelial cell polarization, we determined whether the Na+-H+ exchanger is required for the growth of LLC-PK1 cells. The studies reported here show that there is no obligatory requirement for increased H+ efflux or Na+ entry via the Na+-H+ exchanger for the initiation of cell growth in this epithelial cell line. We used 22Na+ influx, acid extrusion, and intracellular pH determinations to show that onset of cell growth, as measured by DNA content, precedes the activity of the Na+-H+ exchanger in exponentially growing cells, whereas confluent monolayers express Na+-H+ exchanger activity. When confluent cells are replated at low density, Na+-H+ exchanger activity disappears within 8 h in contrast to high-density replated cells. The fact that Na+-H+ exchanger activity is only present in confluent monolayers suggests that the development of tight junctions and polar differentiation play a role in the expression of the Na+-H+ exchanger and that this exchanger is more important to the polar epithelial cell for transepithelial transport than for the maintenance of intracellular pH.
Induction of an increase in mitochondrial matrix enzymes in muscle of iron-deficient rats
Authors: Ohira Y. , Cartier L. J. , Chen M. , Holloszy J. O.
Published: December 2017

Young rats maintained on an iron-deficient diet developed severe anemia and had large decreases in the levels of the iron-containing flavoproteins and cytochromes of the mitochondrial respiratory chain in skeletal muscle. In contrast, the levels of a number of mitochondrial matrix marker enzymes, including citrate synthase, isocitrate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and aspartate aminotransferase, increased in red skeletal muscle but not in white muscle. Phosphocreatine concentration was decreased and inorganic phosphate concentration was increased in soleus muscle frozen in situ. We hypothesize that the increase in mitochondrial matrix enzymes reflects a stimulus to mitochondrial biogenesis in posture-maintaining and weight-bearing red muscle fibers in severely iron-deficient rats. It is our working hypothesis that this stimulus to mitochondrial biogenesis arises from mild activity of the red fibers and is due to the same perturbation in cellular homeostasis that is normally caused by vigorous exercise or hypoxia. In iron deficiency, the stimulus to mitochondrial biogenesis can induce an increase in only those enzymes not prevented from increasing by iron deficiency, resulting in formation of mitochondria of grossly abnormal composition.
(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells
Authors: Liu S. , Jacob R. , Piwnica-Worms D. , Lieberman M.
Published: December 2017

The coupled movements of Na, K, and Cl were studied in cultured chick embryonic heart cells using ion-selective microelectrodes. Movements of K and Cl in response to changes in extracellular [K] ([K]o) showed a furosemide-sensitive coupled process. The movement of Na was then studied. Lowering extracellular [Na] ([Na]o) to 27 mM caused a decrease in intracellular Cl activity (aicl). Upon restoring [Na]o to 143 mM, Cl was taken up against its electrochemical gradient (delta mu Cl). In Cl-free solution, cells lost Na against delta mu Na and simultaneously lost Cl. Upon restoring extracellular [Cl] ([Cl]o), Cl was taken up against delta mu Cl; this was accompanied by an uptake of Na. The Cl uptake was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-insensitive (0.1 mM) but inhibited by removing Nao. Both Cl and Na uptakes were potentiated by raising [K]o from 5.4 to 15 mM, and Na uptake was diminished by lowering [K]o to 1 mM. In all experiments, Cl and Na movements were furosemide (0.3 mM) or bumetanide-sensitive (0.1 mM). Removal of Nao, with resultant depletion of intracellular [Na] ([Na]i), blocked the furosemide or bumetanide-sensitive Cl loss or uptake upon exposure to zero or 133 mM [K]o + SITS (0.1 mM), respectively. These results suggest that cultured heart cells possess an electroneutral (Na + K + 2Cl) cotransport.
Uptake and degradation of natural surfactant by isolated rat granular pneumocytes
Authors: Fisher A. B. , Chander A. , Reicherter J.
Published: December 2017

It has been previously shown that isolated granular pneumocytes internalize and degrade dipalmitoylphosphatidylcholine (DPPC) in synthetic lipid vesicles and reutilize degradation products for phosphatidylcholine (PC) synthesis. In this study, we evaluated the uptake and degradation of radiolabeled natural surfactant (NS) isolated from lung lavage after perfusing isolated rat lungs with [3H]choline. Uptake of NS by isolated granular pneumocytes was increased approximately fourfold compared with synthetic liposomes, suggesting that physical form or a component (e.g., a protein) of NS plays a role in phospholipid uptake by these cells. Uptake was significantly decreased by metabolic inhibitors, indicating an energy requirement for this process. After 2-h incubation, the pattern of radioactivity in cells compared with NS showed a significant decrease in PC and DSPC and increase in free choline, choline phosphate, and CDP-choline. This pattern of metabolism indicates degradation of PC and metabolic reutilization of products. These studies support the hypothesis that alveolar phospholipids are accumulated and reutilized by granular pneumocytes for surfactant synthesis.
Measurement of cytoplasmic free Ca2+ concentration in bovine tracheal smooth muscle using aequorin
Authors: Takuwa Y. , Takuwa N. , Rasmussen H.
Published: December 2017

The cytoplasmic free Ca2+ concentration in bovine tracheal smooth muscle strips was measured using aequorin. Carbachol induces a rapid rise in aequorin luminescence, which reaches a peak within 1-2 min and then falls to a plateau level in 5-6 min. This plateau gradually declines but remains significantly above the base-line value after 2 h. The initial Ca2+ transient is due to Ca2+ mobilization from an intracellular caffeine-sensitive pool. The plateau appears to be due to Ca2+ influx. Histamine or 5-hydroxytryptamine (serotonin) also induce a Ca2+ transient followed by a plateau phase which is lower than that induced by carbachol. The isometric tension generated by either of these two agonists shows a gradual decline in contrast to the sustained plateau seen in the carbachol-induced contraction. Extracellular high K+ induces a dose-dependent increase in aequorin luminescence that is totally due to Ca2+ influx across the plasma membrane and is greatly inhibited by a Ca2+-channel antagonist, nimodipine. These results suggest that two temporally and spatially different Ca2+-dependent mechanisms are involved in carbachol-induced tracheal smooth muscle contraction.
Effect of phorbol ester and calcium ionophore on chloride secretion in canine tracheal epithelium
Authors: Welsh M. J.
Published: December 2017

The control of Cl- secretion was examined by two of the terminal events in the phosphoinositide regulatory pathway: activation of protein kinase C and an increase in cell Ca2+. The phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, stimulated Cl- secretion in both native and cultured monolayers of tracheal epithelium. Approximately 1.5 nM of mucosal PMA was required for half-maximal stimulation. Stimulation was not dependent on prostaglandin production nor was it accompanied by an increase in cellular levels of cAMP. Although the maximal rate of PMA-induced Cl- secretion was less than that induced by cAMP, there was a synergistic effect between PMA and forskolin, an agent that activates adenylate cyclase. The response to PMA was at least partly transient and PMA may also attenuate Cl- secretion under some circumstances. Thus the response to PMA, and presumably protein kinase C activation, may be complex. An increase in cell Ca2+ produced by addition of the Ca2+ ionophore A23187 also stimulated Cl- secretion. However, the effect was at least partly indirect. A23187 enhanced prostaglandin E2 production and the prostaglandin synthesis inhibitor, indomethacin, blocked A23187-induced secretion in native epithelia and attenuated the effect of A23187 in cultured monolayers. These results indicate the presence of a non-cAMP-dependent regulatory pathway capable of controlling Cl- secretion in tracheal epithelium. Activation of protein kinase C may stimulate secretion directly or modulate the response to cAMP. An increase in cell Ca2+ may stimulate secretion at least partly by stimulating endogenous prostaglandin production.