bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncoupling of cardiac cells by doxyl stearic acids specificity and mechanism of action
Authors: Burt J. M.
Published: December 2017

The influence of doxyl stearic acids (DSAs) on gap junctional conductance (gj) between pairs of neonatal rat heart cells was studied. DSAs are spin probes that perturb the membrane at different depths depending on position of the doxyl group on the fatty acyl chain. 16-DSA and 12-DSA rapidly and reversibly reduced gj to unmeasureable levels in a dose- and time-dependent manner. Single channel events observed when gj was low were of the same unitary size as those observed under control conditions. The methyl esters of 16- and 12-DSA, stearic acid itself, and TEMPO, an analogue of the doxyl group that has no fatty acyl chain, had no effect on gj. Protonation of the carboxyl head group (by acidifying the solution) reduced the potency of 16- or 12-DSA. Spontaneous beating activity and action potentials were observed at concentrations of the DSAs 15-20 times that necessary for uncoupling. These results indicate that uncoupling by the DSAs requires the presence of the charged carboxyl group and localized perturbation of the channel at the lipid-channel interface by the doxyl group. Furthermore, they predict that unsaturated free fatty acids, which accumulate during ischemia, may exert their arrhythmogenic effect by reducing gj, and thereby slowing conduction.
Cells in culture from rabbit medullary thick ascending limb of Henle's loop
Authors: Drugge E. D. , Carroll M. A. , McGiff J. C.
Published: December 2017

Freshly isolated cells obtained from the medullary segment of the rabbit thick ascending limb of Henle's loop (mTALH) metabolize arachidonic acid (AA) primarily by the cytochrome P-450 monooxygenase pathway forming several products; a vasorelaxant and an inhibitor of Na+-K+-ATPase have been identified. These studies have been extended to mTALH cells in culture. The ability of cells isolated from 1-mo-old rabbits to grow in culture far surpassed that of cells isolated from adult rabbits, whereas similar cytochrome P-450-dependent AA metabolites were produced by freshly isolated cells from rabbits of both ages. Three-week-old mTALH cultures formed ouabain-sensitive "domes" when grown on plastic surfaces and developed transepithelial voltages (4.7 + 1.2 mV, n = 6) when grown on gas-permeable surfaces. Electron microscopy of the cells showed typical mTALH cell characteristics. The presence of Tamm-Horsfall protein, a surface membrane protein of mTALH cells, in 90-95% of the cells confirmed the homogeneity of the cultures. Although several environmental manipulations were tested, mTALH cells in culture did not produce the same cytochrome P-450-dependent AA metabolites as those produced by mTALH cells before culture. However, a cytochrome P-450-dependent AA metabolite that differs from the AA metabolites formed by freshly isolated mTALH cells was produced by hemin-treated mTALH and heterogenous cell cultures.
Cytosolic free calcium concentration in individual cardiac myocytes in primary culture
Authors: Cheung J. Y. , Tillotson D. L. , Yelamarty R. V. , Scaduto R. C.
Published: December 2017

Cytosolic free Ca2+ concentration, [Ca2+]i, of single isolated Ca2+-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration, [Ca2+]i in quiescent, rod-shaped myocytes was 90 +/- 6 nM. There was no detectable spatial heterogeneity in [Ca2+]i in resting myocytes. Localized regions of [Ca2+]i elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca2+]i (greater than 1 microM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca2+]i but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca2+ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca2+]i in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.
Isoproterenol, DBcAMP, and forskolin inhibit cardiac sodium current
Authors: Ono K. , Kiyosue T. , Arita M.
Published: December 2017

We studied the effects of isoproterenol (ISP), dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and forskolin on the sodium current (INa) of guinea pig ventricular myocytes using the tight-seal, whole cell voltage-clamp method. The extracellular [Na+] [( Na+]o) was decreased to 60 mM by replacing NaCl with sucrose (temperature, 32-33 degrees C). Ionic currents other than Na+ were suppressed using appropriate channel blockers. Depolarizing clamp pulse (duration, 30 ms) was applied at a rate of 0.2 Hz from a holding potential of -80 mV. ISP (1 microM) decreased the peak INa by 34% from 6.1 +/- 1.9 (SD) nA (control) to 4.0 +/- 1.5 nA (n = 7). The inhibition was more prominent at less negative potentials and disappeared in the presence of a beta-blocker (10 microM atenolol). The effects of DBcAMP (1-5 mM) and forskolin (3 microM) mimicked those of ISP and depressed the peak INa reversibly. DBcAMP (5 mM) shifted the inactivation curve of INa [h infinity-membrane potential (Em) relationship] to a hyperpolarizing direction, by 3.4 +/- 0.8 mV (n = 5). These findings suggest that ISP inhibits the cardiac INa+, probably by altering the gating mechanism of the Na+ channel, and that the effect is secondary to the increased levels of intracellular cAMP, with possible acceleration of cAMP-dependent phosphorylation of the channel.
GABA-induced chloride current in rat isolated Purkinje cells
Authors: Kaneda M. , Wakamori M. , Akaike N.
Published: December 2017

Electrical and pharmacological properties of the gamma-aminobutyric acid (GABA)-induced current in the rat isolated cerebellar Purkinje cell bodies were studied using a concentration-jump method, which is termed as a "concentration-clamp" technique. This technique enables the rapid exchange of external solution around the neurons to be perfused internally under a voltage-clamp condition. The peak amplitude of GABA response increased sigmoidally with the increase of the concentration of GABA. The value of the GABA concentration that evokes a half-maximal response (Ka) was 5 X 10(-5) M, and the Hill coefficient was 1.8. The current-voltage relationship for the GABA response showed nonlinearity at membrane potentials more negative than -40 mV. The reversal potential of GABA-evoked current was close to the equilibrium potential of Cl- (ECl), indicating that the current elicited by GABA is carried by Cl-. Both the activation and inactivation phases of GABA-induced Cl- current (ICl) consisted of fast and slow components. These time constants in both phases decreased as the concentration of GABA increased. Strychnine and bicuculline inhibited the GABA-induced ICl in a dose-dependent manner, and the inhibition of the GABA response by bicuculline was competitive. Pentobarbital sodium augmented the GABA response and modified the inactivation phase. The augmentation of the GABA response by pentobarbital was more profound at lower concentrations of GABA and was accompanied by a change in the Hill coefficient from 2 to 1. The properties of the GABA response in cerebellar Purkinje cells were thought to be basically similar to those previously reported in other preparations.
Regulation of synthesis of prostacyclin and HETEs in human endothelial cells
Authors: Ibe B. O. , Falck J. R. , Johnson A. R. , Campbell W. B.
Published: December 2017

Human umbilical endothelial cells in culture synthesize prostacyclin (PGI2), 15-hydroxyeicosatetraenoic acid (15-HETE), and 12-hydroxyeicosatetraenoic acid (12-HETE). The synthesis of these eicosanoids was measured by specific radioimmunoassays after stimulation by arachidonic acid, A23187, bradykinin, melittin, or histamine. Under all conditions, the synthesis of PGI2 paralleled and exceeded the synthesis of 15-HETE and 12-HETE. Indomethacin inhibited arachidonic acid-stimulated PGI2 and 15-HETE synthesis but enhanced 12-HETE synthesis. Meclofenamate gave similar qualitative results. Drugs that act as inhibitors of lipoxygenase in some tissues, such as nordihydroguaiaretic acid (NDGA), caffeic acid, esculin, diethylcarbamazine, quercetin, and 5,8,11,14-eicosatetrayenoic acid (ETYA) were nonspecific in their inhibition of PGI2, 12-HETE, and 15-HETE synthesis. For example, NDGA inhibited arachidonic acid-stimulated release with a 50% inhibitory concentration (IC50) of 0.39 microM for PGI2, 0.25 microM for 15-HETE, and 0.10 microM for 12-HETE. These results show that endothelial cells metabolize both endogenous and exogenous arachidonic acid to PGI2, 15-HETE, and 12-HETE. These data also suggest, based on results with inhibitors, that PGI2 and 15-HETE are products of cyclooxygenase, whereas 12-HETE is produced via a different enzymatic pathway, most likely a lipoxygenase pathway.
Forskolin activates gated Cl- channels in frog skin
Authors: De Wolf I. , Van Driessche W. , Nagel W.
Published: December 2017

We investigated the effect of forskolin on Cl- movements across the isolated epithelium of frog skin. With Cl- on both sides, forskolin (50 mumol/l) increased the transepithelial conductance considerably and elicited significant Cl- secretion. Establishing transepithelial Cl- gradients markedly increased the Cl- currents (ICl). During forskolin treatment, the power density spectra (PDS) of the fluctuation in transepithelial current contained a Lorentzian component that depended on the presence of Cl- in the bathing solutions. Mucosal as well as serosal diphenylamine-2-carboxylic acid (DPC; 1 mmol/l) partially depressed ICl as well as the Lorentzian noise component. Microelectrode recordings from cells involved in transepithelial Na+ absorption showed that forskolin activates gated Cl- channels in a cellular pathway in parallel with the Na+-transporting granulosum cells of the frog skin. The activation of the Cl- -dependent currents and Lorentzian noise was rather variable, and adaptation of the animals to solutions that contained 40 or 60 mmol/l NaCl increased the sensitivity to forskolin. In skins of salt-adapted animals, oxytocin (0.1 U/ml) also slightly activated the Cl- pathway. On the other hand, oxytocin and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; 1 mmol/l) were without effect in control skins.
Retroendocytosis of insulin in a cultured kidney epithelial cell line
Authors: Dahl D. C. , Tsao T. , Duckworth W. C. , Mahoney M. J. , Rabkin R.
Published: December 2017

It has been generally accepted that in renal tubular epithelium endocytosed proteohormones are transported to lysosomes where they undergo complete hydrolysis. En route, as endosomal pH falls, the proteohormone uncouples from the endocytosed membrane binding site, which recycles to the cell surface. However, studies in other tissues have uncovered alternate intracellular pathways for proteins. One such pathway is retroendocytosis (endocytosis then exocytosis). To determine whether a retroendocytotic pathway exists for insulin in renal epithelium, a study was carried out with confluent monolayers of a proximal-like opossum kidney cell line that exhibits receptor-mediated endocytosis of insulin. Cells were preloaded with 125I-labeled insulin (4 X 10(-10) M) for 30 min, surface-bound insulin was then removed by acid washing, and over the next 60 min the release of intracellular radioactivity into the medium was monitored. At 37 degrees C, control cells released on average 7-15% of the intracellular radioactivity as intact insulin [trichloroacetic acid (TCA)-precipitable radioactivity] and approximately 62% as TCA-soluble degradation products. In the presence of 0.1 mM chloroquine (an acidotropic agent) the release of intact insulin increased approximately twofold while degradation fell by nearly one-half. With Sephadex G-50 chromatography we found that the released radioactivity included insulin-size material that increased in the presence of chloroquine. High-performance liquid chromatography revealed that 53 (controls) and 81% (chloroquine treatment) of this latter material consisted of intact insulin. We conclude that, in addition to a major degradative pathway, cultured kidney epithelial cells exhibit a retroendocytotic pathway for insulin. Chloroquine inhibits degradation and appears to divert insulin from the degradative into the retroendocytotic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
Accumulation of some hexoses in adipocytes: properties of the system
Authors: Thompson K. A. , Kleinzeller A.
Published: December 2017

Adipocytes accumulate 2-deoxy-D-glucose (2-DG) against significant chemical gradients (J. Foley and J. Gliemann. Biochim. Biophys. Acta 648: 100-106, 1981). The specificity of this accumulation process was examined. Isolated rat adipocytes incubated at 37 degrees C with 0.1 mM hexoses accumulated free 2-deoxy-D-galactose (2-DGal) or D-galactose (Gal) against steady-state gradients of 5.5 +/- 1.2 and 2.5 +/- 0.7, respectively. Like 2-DG, both 2-DGal and Gal are substrates for phosphorylation. Insulin and insulin-mimetic agents increased steady-state accumulations of Gal, 2-DGal, and free and phosphorylated 2-DG (100 nM insulin increased free 2-DG from 2.1 +/- 0.2 to 6.6 +/- 0.3 mM; external 2-DG = 0.1 mM). Removal of extracellular calcium or sodium or the presence of A23187 or ouabain failed to inhibit 2-DG accumulation. Plasma membrane permeabilization induced by either digitonin or high-voltage discharge produced a loss of cellular 2-DG and 2-DG-phosphate without affecting 3-O-methyl-D-glucose equilibration. The data indicate that neither transmembrane ionic gradients nor intracellular compartmentation suffice as explanations of the mechanism of the accumulation process. The possible role of phosphorylation in the process of hexose accumulation is discussed.
Mechanism of increased erythrocyte membrane fluidity during magnesium deficiency in weanling rats
Authors: Tongyai S. , Rayssiguier Y. , Motta C. , Gueux E. , Maurois P. , Heaton F. W.
Published: December 2017

The erythrocyte membrane was investigated in weanling male rats pair fed with magnesium-deficient and control diets for 8 days. Fluorescence polarization studies revealed a 15% increase in the fluidity of membranes from deficient rats. A similar increase in the fluidity of liposomes indicated that protein was not involved. The change was associated with decreased osmotic fragility of intact erythrocytes; the cells lost their biconcavity and had a flattened appearance with surface irregularities. Analysis of the membranes showed decreased amounts of magnesium, cholesterol, and sphingomyelin in the deficient group. The reduced ratios of cholesterol to phospholipid and sphingomyelin to phosphatidylcholine were consistent with the increased fluidity. Addition of physiological amounts of magnesium to the medium rigidified membranes incubated in tris(hydroxymethyl)-aminomethane buffer, and this was prevented by the presence of EDTA. Cross-incubation experiments with erythrocyte ghosts and plasma from the two groups of rats showed that magnesium-deficient plasma increased the fluidity of control ghosts and control plasma rigidified ghosts from magnesium-deficient rats. Addition of sufficient magnesium chloride to raise the magnesium content of deficient plasma to normal had no significant effect. These results show that the increased fluidity of the erythrocyte membrane in magnesium deficiency is due to physicochemical exchange with the plasma. Although magnesium can directly influence membrane fluidity, the change during its deficiency in vivo is mainly mediated indirectly via disturbances in lipid metabolism.
An ATP-sensitive conductance in cultured smooth muscle cells from pregnant rat myometrium
Authors: Honore E. , Martin C. , Mironneau C. , Mironneau J.
Published: December 2017

The whole cell voltage-clamp technique was used to study the effects of extracellular ATP in cultured smooth muscle cells isolated from pregnant rat myometrium. An inward current was elicited by ATP (IATP) in cells held at -70 mV under voltage clamp. The amplitude of IATP was reduced by estrogen pretreatment and by the end of pregnancy. IATP not only did not undergo any desensitization but showed facilitation. The current-voltage relationship of IATP was linear and reversed close to 0 mV. Changing the sodium electrochemical gradient by decreasing extracellular or intracellular sodium resulted in a linear relationship between the reversal potential of IATP and Na equilibrium potential that, however, differed from the predicted curve for a purely sodium conductance. The conductance activated by ATP was monovalent cation selective with little discrimination between potassium, cesium, and sodium ions. IATP was depressed by divalent cations, and the rank order of potency was Co greater than Mg greater than Ca greater than Ba, suggesting that the free-acid form of ATP was the effective ligand. Adenosine, AMP, and ADP were ineffective in eliciting IATP, whereas ATP gamma S and alpha,beta-methylene ATP were capable of mimicking the effects of ATP, although they were less potent. These results are consistent with the free-acid form of ATP activating a monovalent cation-selective and estrogen-sensitive conductance in myometrium.
Contractile force and intracellular Ca2+ during relaxation of canine tracheal smooth muscle
Authors: Gunst S. J. , Bandyopadhyay S.
Published: December 2017

Muscle strips loaded with the Ca2+ indicator aequorin were studied in vitro to determine the effects of inhibitory stimuli on force and cytosolic free Ca2+. In muscles contracted isometrically with acetylcholine (ACh), 5-hydroxytryptamine (5-HT), carbachol, decreases in muscle force caused by isoproterenol (10(-5) M) or forskolin (10(-5) M) were accompanied by proportional decreases in aequorin luminescence. A similar relationship between decreases in muscle force and aequorin luminescence was observed when muscles were relaxed by stimulating Na+-K+-ATPase activity. These results suggest that the Ca2+ sensitivity of contractile proteins was not decreased during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent relaxation. However, aequorin luminescence did not decrease when muscles contracted by K+ depolarization were relaxed with isoproterenol. Incubation of muscles in forskolin depressed increases in both force and aequorin luminescence in response to 5-HT or ACh. Incubation of muscles in isoproterenol had a similar effect on responses to 5-HT but depressed increases in force without depressing increases in luminescence in response to ACh. Results indicate that under most conditions the reduction of cytosolic Ca2+ plays an important role in the cAMP-dependent relaxation of canine tracheal smooth muscle.
Simulated ischemia and intracellular pH in isolated ventricular muscle
Authors: Vanheel B. , Leybaert L. , De Hemptinne A. , Leusen I.
Published: December 2017

Isolated guinea pig papillary muscles were subjected to an in vitro model of ischemia, consisting of superfusion arrest and immersion in paraffin oil, which results in restriction of substrate supply and metabolite washout. Intracellular pH (pHi) and surface pH (pHs) were measured with glass microelectrodes. Contractile force declined to 82% of the pre-“ischemic” value after 2 min and to 37% of the control value after 10 min. In addition, a shortening of the time to peak and duration of contraction was noted. The rate of force development decreased later than the rate of relaxation. After 10 min, pHi was acidified on average 0.08 pH unit, which is about one-third of the measured pHs change. Tripling the ischemic pHi change by reduction of the intracellular buffering power only slightly increased the rate of tension decline. Experimental pHi changes of similar magnitude, induced during normal superfusion, had a smaller effect on contractile force and failed to reproduce the characteristic changes in time course of the contraction. It is concluded that, in our condition of simulated ischemia, the intracellular acidification cannot account fully for the rapid decline in contractility.
Computer analysis reveals changes in renal Na+-glucose cotransporter in diabetic rats
Authors: Blank M. E. , Bode F. , Baumann K. , Diedrich D. F.
Published: December 2017

A novel, computer-assisted program was developed to analyze the time course of Na+-glucose cotransport by rat renal cortical brush-border membrane vesicles (BBMV). Transporter characteristics can be measured, which routine kinetic analyses fail to distinguish: cotransporter membrane density is derived from the picomoles of D-glucose bound per milligram of protein. Binding is stereospecific, blocked by phlorizin, and supported equally well by Na+ or K+ (but not Cs+). Quasi-first-order influx and efflux rate constants for the composite Na+-driven influx and the (presumed) Na+-independent efflux processes were highly dependent on glucose concentration. Either two Na+-glucose transporters exist in proximal tubules or a single mechanism abruptly changes rate when glucose falls to low levels. The major operation mode is slow, has a high capacity but low affinity, and may have a 2 Na+:2 glucose stoichiometry (Hill coefficient is unity). The minor system is a fast, smaller-capacity, higher-affinity operation with a 2 Na+:1 glucose stoichiometry that was not distinguishable when the same data were analyzed in conventional kinetic plots. Results with streptozocin-induced diabetic rats illustrate the method's utility. Low-glucose-affinity cotransporters were upregulated in hyperglycemic, but not in cachectic, ketoacidotic animals. Rate constants, especially for efflux, were decreased in diabetes.
Colchicine-induced redistribution of an apical membrane glycoprotein (gp330) in proximal tubules
Authors: Gutmann E. J. , Niles J. L. , McCluskey R. T. , Brown D.
Published: December 2017

Factors governing the selective, polarized insertion of membrane proteins are poorly understood, but some studies have suggested that microtubules are involved in the generation and maintenance of cell polarity. We have examined by immunocytochemistry the effect of the microtubule-disrupting agent, colchicine, on the cellular distribution of an endogenous glycoprotein, gp330, which is normally inserted only into the apical plasma membrane of proximal tubule epithelial cells. In control rats, gp330 was localized in the brush border and in apical invaginations and vesicles. Six hours after injection of colchicine, however, vesicles containing gp330 were dispersed throughout the entire cytoplasm of the cell. Many vesicles were packed into basolateral infoldings, close to the plasma membrane, but there was no significant insertion of gp330 into the basolateral membrane. When rabbit anti-gp330 antiserum was injected intravenously into colchicine-treated rats, immune complexes appeared in the glomerular basement membrane but could not be detected in peritubular basement membranes. This supports the conclusion that colchicine treatment does not result in the insertion of gp330 into the basolateral plasma membrane of proximal tubule cells. Our results indicate that although microtubules are involved in the accumulation of gp330-containing vesicles at the apical pole of the cell, other factors must be required for fusion with the plasma membrane to occur.
Forskolin and caffeine induce Ca2+ release from intracellular stores in rabbit aorta
Authors: Hai C. M. , Phair R. D.
Published: December 2017

We combined techniques of 45Ca efflux and computer-assisted kinetic analysis to investigate the effects of forskolin and caffeine on intracellular Ca2+ metabolism in intact rabbit aortic segments. When either 1 microM forskolin or 10 mM caffeine was present during the 45Ca load and efflux, the amount of 45Ca released from the tissue was reduced during the interval between 50 and 250 min of efflux. In contrast caffeine but not forskolin induced an acute increase in 45Ca efflux when added to the perfusion medium after 1 h of efflux in physiological salt solution. Preincubation with caffeine abolished phenylephrine-stimulated 45Ca release, but preincubation with forskolin had no effect. Kinetic analysis of these data indicated that caffeine reduced the Ca2+ content of the same intracellular compartment depleted by phenylephrine, whereas forskolin depleted a different intracellular Ca2+ store. Forskolin-induced depletion of an intracellular Ca2+ store was surprising in light of current evidence suggesting adenosine 3',5'-cyclic monophosphate stimulation of sarcoplasmic reticular Ca2+ uptake, but hypotheses that included only this mechanism are inconsistent with our findings.
Convergence of cAMP and phosphoinositide pathways during rat parotid secretion
Authors: McKinney J. S. , Desole M. S. , Rubin R. P.
Published: December 2017

Rat parotid acinar cells were employed to investigate the mechanism by which receptor agonists that activate the phosphoinositide pathway enhance the stimulatory effects of adenosine 3',5'-cyclic monophosphate (cAMP) on amylase secretion. Norepinephrine (NE), which activates both alpha- and beta-adrenoceptors, evoked a secretory response that was greater than the sum of the responses obtained when NE was employed as a beta-agonist (in the presence of prazosin) and as an alpha-agonist (in the presence of propranolol). The enhancement of amylase secretion induced by NE was accompanied by an augmented rise in Ca2+ influx, as determined by fura-2 analysis. NE-induced cAMP production was comparable to that evoked by NE as a beta-agonist, and the accumulation of [3H]inositol 1,4,5-trisphosphate (IP3) evoked by NE was comparable to that elicited by NE as an alpha-agonist. The beta-adrenoceptor agonist isoproterenol potentiated the rise in cytosolic Ca2+ elicited by the muscarinic agonist carbachol, while possessing no stimulatory effect of its own. Isoproterenol had no effect on carbachol-induced stimulation of [3H]IP3 or 1,3,4,5-[3H]inositol tetrakisphosphate accumulation. Ionomycin and dibutyryl cAMP in combination produced a similar enhancing effect on the Ca2+ signal and amylase release as adrenergic and muscarinic receptor agonists. These results suggest that the synergism between the phosphoinositide and cAMP-signaling systems in parotid cells resides in enhanced Ca2+ availability.
Modulation of bioelectric properties across alveolar type II cells by substratum
Authors: Cott G. R.
Published: December 2017

Rat alveolar type II cells were cultured on collagen-coated filters (CCF) and human amnionic basement membrane (ABM) to determine the effect of culture substratum on the development of monolayer bioelectric properties. Monolayers cultured on both substrata rapidly developed bioelectric properties with similar time courses, monolayer capacitance values (approximately 1 muF/cm2), current-voltage relationships, and responses to stimulants and inhibitors of active ion transport. Increasing seeding densities tended to increase monolayer bioelectric properties regardless of culture substratum. Monolayers cultured on ABM had higher resistance values (491 vs. 291 omega.cm2) and lower short-circuit currents (2.85 vs. 4.51 muA/cm2) than monolayers with similar cell densities cultured on CCF. These differences in monolayer bioelectric properties were not due to differences in substratum resistance or capacitance effects. The relationships between monolayer bioelectric properties were also affected by the culture substratum. In additional experiments, cells cultured on contracted gels formed monolayers with high short-circuit currents (9.25 muA/cm2). Cell morphology varied depending on the culture substratum, with cells cultured on contracted gels appearing the most cuboidal, whereas the flattest and most attenuated cells were those cultured on ABM. On the basis of these observations, we conclude that culture substratum significantly affects the development of bioelectric properties across alveolar type II cell monolayers. In vivo the bioelectric properties across the alveolar epithelium may also vary with changes in cellular substratum or cell density (e.g., after acute lung injury) and possibly with cell morphology (e.g., alveolar type I vs. alveolar type II cells).
Stimulation of glucose transport by insulin and norepinephrine in isolated rat brown adipocytes
Authors: Marette A. , Bukowiecki L. J.
Published: December 2017

The effects of insulin and norepinephrine on glucose transport, glucose uptake, and cell respiration were investigated in isolated rat brown adipocytes. Glucose transport and uptake were determined using [U-14C]-D-glucose and 2-deoxy-[1,2-3H]-D-glucose, respectively. Brown adipocyte respiration was measured polarographically. Dose-response experiments revealed that insulin stimulated D-glucose transport and 2-deoxyglucose uptake between 10(-11) and 10(-7) M with a maximal four- to sixfold stimulation. In the absence of insulin, norepinephrine concentrations ranging from 10(-7) to 10(-7) M also enhanced glucose transport and uptake with a maximal two- to fourfold stimulation. Experiments with alpha- and beta-adrenergic agonists and antagonists showed that the effect of norepinephrine was predominantly mediated via beta-adrenergic pathways. Dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine also increased glucose transport, suggesting that the effects of norepinephrine are cyclic AMP dependent. Moreover, norepinephrine (10(-8) M) enhanced insulin sensitivity for glucose transport [half-maximum velocity constant (1/2 V max)] but failed to potentiate insulin responsiveness (Vmax). On the other hand, insulin (10(-9) M) had no effect on basal respiration but rapidly inhibited the calorigenic effect of norepinephrine (10(-7) M) by greater than 50%. These results demonstrate that 1) in the absence of insulin, physiological concentrations of norepinephrine stimulate glucose transport via beta-adrenergic pathways, 2) the neurohormone synergistically potentiates brown adipocyte submaximal insulin responses for glucose transport, and 3) insulin counteracts the effects of norepinephrine on brown adipocyte thermogenesis despite the fact that both hormones enhance glucose uptake.
Glycogen metabolism during tension generation and maintenance in vascular smooth muscle
Authors: Lynch R. M. , Kuettner C. P. , Paul R. J.
Published: December 2017

To study the regulation of glycogen utilization in vascular smooth muscle, we measured the content of glycogen and glucose 6-phosphate and the activity of the glycogen phosphorylase and glycogen debrancher enzymes in porcine carotid artery. During active contraction, the rates of glycogen phosphorylase and glycogenolysis were as high as expected. Despite this, glycogen content did not decrease to less than approximately 50% of control levels even after sustained contractions. The activity of glycogen debrancher enzyme was found to be limiting glycogen utilization at this point. Although glycogenolysis is closely coordinated with increases in oxidative metabolism concomitant with active contraction, the maximal level of tension obtained after stimulation was not substantially reduced under conditions where glycogen debrancher enzyme was limiting glycogen utilization. On the other hand, the rate of tension generation was increased in these tissues. Thus glycogen utilization is not necessary for maximal force generation per se, but may influence other muscle contractile properties. Finally, during steady-state tension maintenance, glycogen utilization is likely to be regulated by the intracellular concentrations of metabolic intermediates (glucose, glucose 6-phosphate), as it is in skeletal muscle.
A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes
Authors: Geering K. , Theulaz I. , Verrey F. , Hauptle M. T. , Rossier B. C.
Published: December 2017

In all cellular systems studied so far, the catalytic alpha- and the glycosylated beta-subunit of Na+-K+-ATPase are coordinately synthesized and are assembled into stoichiometric alpha, beta-complexes. In contrast to these data, in this study we show that the fully grown oocyte of Xenopus laevis synthesizes much less beta-subunit than alpha-subunit. The alpha-subunit produced in excess over the beta-subunit is membrane associated but highly trypsin sensitive and can be compared with the immature alpha-subunit population identified in epithelial cells immediately after synthesis (K. Geering, J. P. Kraehenbuhl, and B.C. Rossier, J. Cell Biol. 105: 2613-2619, 1987). The Xenopus oocyte thus turns out to be a unique system to study the functional role of the beta-subunit. Injection of beta-subunit-specific mRNA transcribed in vitro from a beta-cDNA clone (derived from Xenopus kidney, A6 cells) into oocytes results in translation of a glycosylated beta-subunit. The synthesis of this exogenous beta-subunit increases significantly the proportion of trypsin-resistant oocyte alpha-subunits able to perform cation-dependent conformational changes. In addition, 25-65% more ouabian binding sites are expressed at the plasma membrane in beta-mRNA-injected oocytes. In contrast, newly synthesized alpha-subunit translated after injection of size-fractionated mRNA enriched in alpha-mRNA remains trypsin sensitive as the oocyte alpha-subunit. These data suggest that association of the beta-subunit to the alpha-subunit provokes a structural rearrangement of the alpha-subunit that might be a first step toward the functional maturation of the Na+-K+-ATPase and its expression at the plasma membrane.
Effects of sulfonamides on a metabolite-regulated ATPi-sensitive K+ channel in rat pancreatic B-cells
Authors: Gillis K. D. , Gee W. M. , Hammoud A. , McDaniel M. L. , Falke L. C. , Misler S.
Published: December 2017

Intracellular ATP (ATPi)-sensitive K+ [K+(ATP)] channels are now a recognized site of action of clinically useful hypoglycemic and hyperglycemic sulfonamides. We have further examined the action of these agents on single K+ channels in rat pancreatic B-cells 1) Tolbutamide and glyburide, two hypoglycemic sulfonylureas which decrease K+(ATP) channel activity in the cell-attached patch, affect the kinetics of K+(ATP) channel in a manner similar to glucose. They shorten the duration of the “burst,” or cluster of open channel events, while lengthening the intervals between bursts. 2) The hyperglycemic vasodilator diazoxide increases mean K+(ATP) channel activity in the cell-attached patch as well as in the inside-out excised patch exposed to ATPi. It appears to lengthen channel bursts and shorten the intervals between them. Two structurally similar diuretics, hydrochlorothiazide and furosemide, which have mild hyperglycemic effects, do not increase K+(ATP) channel activity even at clinically toxic concentrations. 3) Neither the sulfonylureas nor diazoxide directly affect the activity of single delayed rectifier K+ channels or single calcium and voltage-activated K+ channels in normal B-cells.
Distinct epitopes on amiloride
Authors: Kleyman T. R. , Kraehenbuhl J. P. , Rossier B. C. , Cragoe E. J. , Warnock D. G.
Published: December 2017

Most Na(+)-selective transport proteins are inhibited by the drug amiloride. Studies using amiloride analogues suggest that specific regions of amiloride might participate in binding to receptors on these transport proteins. To determine whether certain domains of this drug are recognized as distinct epitopes, amiloride was coupled to albumin through either its C-5 NH2-group on the pyrazine ring or through a terminal NH2-group of the guanidino moiety, and antibodies were raised against these amiloride-albumin conjugates. Studies of antibody binding to amiloride analogues identified the 3,5-diaminopyrazinyl, the guanidinocarbonyl, and the C-6 halo moieties as distinct epitopes, although the antibodies required the presence of both the 3,5-diaminopyrazinyl as well as the guanidinocarbonyl moiety for binding.
Copper transport from ceruloplasmin: characterization of the cellular uptake mechanism
Authors: Percival S. S. , Harris E. D.
Published: December 2017

Copper uptake from 67Cu-labeled ceruloplasmin (67CuCp) was studied in K-562 cells, a human erythroleukemic cell line. 67CuCp was prepared by an ascorbate-catalyzed exchange of recrystallized ceruloplasmin with 67CuCl2. The labeled protein was treated with Chelex-100 and gel filtration to ensure that 67Cu was tightly bound to the structure. 67CuCp bound specifically to the K-562 cells at 4 degrees C. The binding was linear with protein in the range of 200-800 nM and in the presence of 3% albumin. In this concentration range, 67CuCl2 showed no binding that could be interpreted as specific; 80-90% of the cell-bound 67Cu was removed by washing the cells with acid buffer. When binding was attempted at 37 degrees C, a significant fraction of the 67Cu resisted acid washing and with time accumulated in the cells. Fractionating the cytosolic components on Percoll gradients located the 67Cu in buoyant fractions of densities 1.030-1.05, with a peak at 1.035. Repeating the experiment with 125I-labeled ceruloplasmin failed to localize any 125I label in Percoll fractions; very little 125I was detected in the cytosol. Double-labeled 67Cu-125I-ceruloplasmin confirmed that copper and not the protein moiety of ceruloplasmin was taken up by the cells. The uptake reaction was inhibited by 1 mM bathocuproine sulfonate and by 1 mM sodium iproniazid. Ascorbate (100 microM) strongly stimulated uptake. These studies provide evidence that K-562 cells are able to extract copper atoms from ceruloplasmin and transport the copper to the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of GTP gamma S on insulin binding and tyrosine phosphorylation in liver membranes and L6 muscle cells
Authors: Burdett E. , Mills G. B. , Klip A.
Published: December 2017

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a specific activator of G proteins, did not change the Kd nor total binding of [125I]insulin in plasma membranes from rat liver. Insulin did not alter GTP gamma 35S binding nor polypeptide ADP ribosylation in crude and plasma membranes catalyzed either intrinsically or by cholera toxin. In L6 muscle cells, insulin caused tyrosine phosphorylation of a polypeptide of Mr 160,000. Cell electroporation enabled testing of G protein action in this cellular system. Phosphorylation of the Mr 160,000 polypeptide in these permeabilized cells was insulin and ATP dependent but other small molecules or ionic gradients were not essential. The reaction could not be mimicked by the G protein agonist GTP gamma S nor inhibited by the G protein antagonist guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). However, GTP gamma S effectively decreased insulin-mediated phosphorylation of this polypeptide. This suggests that the tyrosine kinase activity of the insulin receptor can be modulated by G protein agonists. It is concluded that cross talk between the insulin receptor and G proteins could not be demonstrated in isolated membranes by strategies that detect interactions between beta-adrenergic receptors and G proteins. In contrast, in permeabilized cells, G protein-mediated regulation of the insulin receptor kinase activity could be detected.
Age, anatomic site, and the replication and differentiation of adipocyte precursors
Authors: Kirkland J. L. , Hollenberg C. H. , Gillon W. S.
Published: December 2017

The effects of donor age and anatomic site on cellular replication and differentiation were studied in adipocyte precursors cloned from epididymal and perirenal depots of young, middle-aged, and senescent rats. As animals aged from 3 to 29 mo, there was a progressive reduction in the proportion of cells capable of extensive replication in both depots. An inverse relation between clonal capacity for replication and differentiation was found. This relation was affected by donor site but not age. Aging was, however, associated with a reduction in the frequency of clones capable of full differentiation into cells with single, large, central lipid inclusions. Hence, age and donor site may affect adipocyte precursor replication and differentiation by different mechanisms.
Cotransport of sodium and chloride by the adult mammalian choroid plexus
Authors: Johanson C. E. , Sweeney S. M. , Parmelee J. T. , Epstein M. H.
Published: December 2017

Cerebrospinal fluid formation stems primarily from the transport of Na and Cl in choroid plexus (CP). To characterize properties and modulation of choroidal transporters, we tested diuretics and other agents for ability to alter ion transport in vitro. Adult Sprague-Dawley rats were the source of CPs preincubated with drug for 20 min and then transferred to cerebrospinal fluid (CSF) medium containing 22Na or 36Cl with [3H]mannitol (extracellular correction). Complete base-line curves were established for cellular uptake of Na and Cl at 37 degrees C. The half-maximal uptake occurred at 12 s, so it was used to assess drug effects on rate of transport (nmol Na or Cl/mg CP). Bumetanide (10(-5) and 10(-4) M) decreased uptake of Na and Cl with maximal inhibition (up to 45%) at 10(-5) M. Another cotransport inhibitor, furosemide (10(-4) M), reduced transport of Na by 25% and Cl by 33%. However, acetazolamide (10(-4) M) and atriopeptin III (10(-7) M) significantly lowered uptake of Na (but not Cl), suggesting effect(s) other than on cotransport. The disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 10(-4) M), known to inhibit Cl-HCO3 exchange, substantially reduced the transport of 36Cl. Bumetanide plus DIDS (both 10(-4) M) caused additive inhibition of 90% of Cl uptake, which provides strong evidence for the existence of both cotransport and antiport Cl carriers. Overall, this in vitro analysis, uncomplicated by variables of blood flow and neural tone, indicates the presence in rat CP of the cotransport of Na and Cl in addition to the established Na-H and Cl-HCO3 exchangers.
Effect of hyperosmotic challenge on basolateral membrane potential in rabbit urinary bladder
Authors: Donaldson P. J. , Lewis S. A.
Published: December 2017

In the rabbit urinary bladder, serosal hyperosmotic challenge (SHOC) with either 33 mM NaCl or 66 mM mannitol caused basolateral membrane potential (Vbl) to initially depolarize from -52.6 +/- 1.6 to -48.4 +/- 1.4 mV, followed by a recovery of Vbl to -57.5 +/- 1.3 mV after 13.7 +/- 1.0 min. The voltage recovery was dependent on both serosal HCO3- and Cl-, and in the absence of both, Vbl depolarized to -11.6 +/- 1.5 mV and the ratio of apical-to-basolateral resistance (Ra/Rbl) decreased from 21.0 +/- 3.4 to 8.3 +/- 3.1. This decrease in Ra/Rbl and consequent depolarization of Vbl is caused by a decrease in basolateral K+ conductance. Replacement of serosal Cl- with NO3- or SCN- followed by SHOC caused a sustained depolarization of Vbl to -32.5 +/- 4.4 and -40.9 +/- 0.9 mV, respectively. However, when Br- was used to replace Cl-, voltage recovery occurred but was slowed (24.0 +/- 2.7 min) and reduced in magnitude (-47.5 +/- 3.5 mV). Addition of amiloride (1 mM) or niflumic acid (100 microM), but not bumetanide (1 microM), to the serosal bathing solution inhibited voltage recovery causing Vbl to depolarize to -36.3 +/- 2.6 and -41.5 +/- 4.5 mV, respectively. Serosal addition of ouabain after SHOC caused Vbl to depolarize by 10.8 +/- 0.9 mV in 2 min. We speculate that the SHOC-induced initial depolarization of Vbl is a loss of Ba2(+)-sensitive K+ conductance caused by cell shrinkage. The subsequent repolarization/hyperpolarization of Vbl is caused by an enhanced basolateral membrane Na+ pump current and a reappearance of the Ba2(+)-sensitive K+ conductance. The parallel operation of Na(+)-H+ and Cl(-)-HCO3- exchanges will then supply Na+ for the pump current and, via cellular accumulation of Na+, K+, and Cl-, might result in a partial recovery of cell volume and thus Ba2(+)-sensitive K+ conductance.
Chloride channels in the apical membrane of a distal nephron A6 cell line
Authors: Marunaka Y. , Eaton D. C.
Published: December 2017

In this report, single-channel recording methods were used to determine whether there are Cl- conductive pathways in the apical membrane of cultured renal distal nephron cells (A6). Two different types of single Cl- channels were observed. In cell-attached patches, one had a unit conductance of 3 pS, whereas the unit conductance of the other was 8 pS. In cell-attached patches, the currents associated with the 3-pS Cl- channel outwardly rectified, whereas the current voltage relationship for the 8-pS Cl-channel was linear. The 3-pS Cl- channel has one open and one closed state; the 8-pS Cl- channel has one open and two closed states. The open probability of the 3-pS Cl- channel was voltage dependent (increasing with depolarization of the membrane) but even at very depolarized potentials (+140 mV) remained small (always less than 0.1). On the other hand, the open probability of the 8-pS Cl- channel was large (approximately 0.8) and voltage independent. The closing rate of the 3-pS Cl- channel was decreased when the patch membrane was depolarized, whereas the opening rate was increased. In contrast, the closing rate of the 8-pS Cl- channel decreased with depolarization, but the opening rates were voltage independent. The outward rectification of the 3-pS channel was markedly reduced in inside-out patches when high calcium concentrations (10-800 microM) were present on the intracellular surface. The open probability of the 3-pS Cl- channel is increased by membrane permeable analogues of adenosine 3',5'-cyclic monophosphate primarily by decreasing the mean closed time.
Steady-state pHi, buffering power, and effect of CO2 in a smooth muscle-like cell line
Authors: Putnam R. W. , Grubbs R. D.
Published: December 2017

Intracellular pH (pHi) was studied in the smooth muscle-like cell line, BC3H-1, using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The initial pHi measured in 20 mM Na N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid-buffered medium [NHB; external pH (pHo) 7.4, 37 degrees C] was 6.89 +/- 0.01 (n = 178). pHi was affected by changes in external pHo, pHi changing by approximately 70% of the change in pHo. The intrinsic buffering power (beta int) of these cells, measured either with NH4Cl or Na propionate pulses, is low for muscle cells, averaging approximately 10 mM/pH unit. Steady-state pHi of BC3H-1 cells in NHB acidified reversibly on exposure to 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1.4 +/- 0.3 x 10(-4) pH/s), 1 mM amiloride (2.0 +/- 0.7 x 10(-4) pH/s), or Na-free solution (8.3 +/- 2.4 x 10(-4) pH/s) and alkalinized upon exposure to Cl-free solutions (9.7 +/- 2.2 x 10(-4) pH/s). Exposure of BC3H-1 cells to CO2-HCO3-buffered solutions resulted in a transient acidification followed by an alkalinization of 0.3-0.4 pH unit to a new steady-state pHi of 7.27 +/- 0.01 (n = 65). This new steady-state pHi acidified very slowly upon exposure to 1 mM amiloride (0.3 +/- 0.1 x 10(-4) pH/s), acidified more rapidly upon exposure to 0.5 mM DIDS (5.9 +/- 0.6 x 10(-4) pH/s) or Na-free solutions (9.8 +/- 1.0 x 10(-4) pH/s), and alkalinized on exposure to Cl-free solutions (24.5 +/- 1.3 x 10(-4) pH/s).(ABSTRACT TRUNCATED AT 250 WORDS)