bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of theophylline, verapamil, and diltiazem on hamster diaphragm muscle force in vitro
Authors: Esau S. A.
Published: December 2017

The effect of the calcium channel blocker verapamil (5 microM) or diltiazem (5 microM) on force production and resting membrane potential (Em) was studied in the hamster diaphragm muscle. The calcium channel blockers decreased force at high frequencies of stimulation and decreased Em by 3 +/- 1 mV. The interaction of theophylline and the calcium channel blockers was also studied. When given simultaneously, the calcium channel blockers prevented the increase in force at high frequencies with theophylline at 1 mM and at all frequencies with theophylline at 0.55 mM. There was no change in Em when theophylline and calcium channel blockers were given together. Similar results were obtained by removing external calcium. The decrease in force produced by verapamil was not reversed by subsequent addition of theophylline. Theophylline did reverse the decrease in force produced by diltiazem. Verapamil reversed the increase in force at high frequencies produced by theophylline. Diltiazem reversed the force increase at low frequencies as well. These studies suggest that extracellular calcium is important in skeletal muscle contractility especially at high frequencies. Extracellular calcium is also important for the potentiation of force and the hyperpolarization of the Em produced by theophylline. The mechanism by which extracellular calcium contributes to these actions of theophylline is not known.
Modulation of tight junction formation in clone 4 MDCK cells by fatty acid supplementation
Authors: Schneeberger E. E. , Lynch R. D. , Kelly C. A. , Rabito C. A.
Published: December 2017

Clone 4 MDCK cells, which generate a transepithelial electrical resistance (TER) of greater than 2,000 omega.cm2, were used to examine the role of membrane lipids in the barrier function of tight junctions. Phospholipid acyl groups were modified by supplementing cells grown in serum-free medium with 18:1(n-9), 18:3(n-3), or 18:3(n-6) complexed to albumin. Although both of these polyunsaturated fatty acids depress the melting point of membrane phospholipids, only 18:3(n-6) contributes significantly to eicosanoid production. Saturation indices of the phospholipids of cells supplemented with albumin alone, 18:1(n-9), 18:3(n-3), or 18:3(n-6) were 0.77, 0.78, 1.81, and 1.65, respectively. After trypsinization or removal of Ca2+, cells supplemented with 18:3(n-6) required longer periods of time to reestablish TER than did nonsupplemented cells or those incubated with 18:3(n-3) or 18:1(n-9). In contrast to MDCK strains I and II, clone 4 MDCK cells required continuous protein synthesis not only to reseal preexisting junctions after the addition of Ca2+ to Ca2+-depleted monolayers, but also to maintain steady-state TER. The rate of decay of TER in the presence of 1 microgram/ml of cycloheximide was 1.5 times greater in cells supplemented with either of the two 18:3 isomers than it was in nonsupplemented controls or in cells supplemented with 18:1(n-9). No significant difference was observed in the steady-state TER or selectivity of the tight junctions after fatty acid supplementation. These results suggest that there is a change in the dynamics of junction formation, rather than an alteration in their intrinsic properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Ca2+ release from sarcoplasmic reticulum of skinned fast- and slow-twitch muscle fibers
Authors: Salviati G. , Volpe P.
Published: December 2017

We have performed a comparative study of Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned fibers from rabbit fast- and slow-twitch skeletal muscle. Ca2+ fluxes have been indirectly monitored by following either tension development or the inhibition of net Ca2+-loading rate by a light-scattering method. Several drugs (Ca2+-release modulators) have been used to either trigger or block Ca2+ release. Our results indicate that caffeine, doxorubicin, and ryanodine activate Ca2+ release, whereas ruthenium red blocks Ca2+ release from both fast- and slow-twitch skinned fibers. Caffeine has greater affinity for slow SR, whereas doxorubicin, ruthenium red, and ryanodine have greater affinity for fast SR. Our results indicate that Ca2+-release mechanisms in fast and slow SR are homologous but not identical and that differences in twitch-contraction time might be also related to the inherent properties of the Ca2+-release mechanism.
Dynamic NMR measurement of volume regulatory changes in Amphiuma RBC Na+ content
Authors: Anderson S. E. , Adorante J. S. , Cala P. M.
Published: December 2017

23Na nuclear magnetic resonance (NMR) and conventional chemical methods were employed to measure Na+ fluxes in Amphiuma red blood cells (RBC) during volume regulation. Paramagnetic shift reagents [dysprosium triethylenetetraminehexaacetic acid (DyTTHA) and dysprosium tripolyphosphate (Dy(TPP)2)] were used to alter extracellular Na+ magnetic resonance. Data are presented describing 23Na resonance dependence on shift reagent, sodium and calcium concentration. We confirmed that the shift reagents neither enter the cells nor alter intracellular Na+, K+, and Cl-concentrations under control conditions when extracellular calcium was maintained greater than 0.5 mM. We also confirmed that the shift reagent complexes chelate calcium [Dy(TPP)2 much more so than DyTTHA] and that their toxic effects could be alleviated by adjusting calcium in the cell's suspension medium to control levels. In parallel experiments, where volume-activated Na+ fluxes ranged from 0.3 to 3 mmol Na+/kg dry cell solid (DCS) x minute in cells containing from 30 to 150 mmol Na+/kg DCS, changes in intracellular sodium measured by 32Na NMR were within 4% of those measured by conventional destructive methods. Finally, we present data that are consistent with the interpretation that 6 mmol Na+/kg DCS plus 16% of intracellular Na+ is NMR invisible.
A bioenergetic model of gossypol action: effects of gossypol on adult rat spermatogenic cells
Authors: Reyes J. , Borriero L. , Benos D. J.
Published: December 2017

Rat spermatogenic cells have glycolytic rates of 124 +/- 36 nmol lactate.mg protein-1.h-1. Maximum glycolytic rates in the absence of oxidative phosphorylation do not exceed the basal rates by greater than 40%. From the rates of oxygen consumption and lactate production it can be calculated that less than 8% of the total ATP produced by spermatogenic cells is provided by glycolysis. These properties of spermatogenic cells would render them highly susceptible to cytotoxicity due to the action of agents like gossypol that impair mitochondrial oxidative phosphorylation or to conditions that produce testicular hypoxia. The main effect of gossypol on spermatogenic cell energy metabolism is to uncouple mitochondrial oxidative phosphorylation. In the absence of glucose, this uncoupling action of gossypol would change the NAD+/NADH redox coupling and, consequently, could modify metabolic fluxes through the lactic dehydrogenase catalyzed reaction.
Ouabain-resistant, amiloride-sensitive Na+-K+ pumping activity and morphological changes are inducible
Authors: Epstein J. A. , Lechene C.
Published: December 2017

The functional expression of a ouabain-resistance gene has been studied in a transfected cell line, OR-6 cells. These cells possess a ouabain-inhibitable, amiloride-resistant Na+-K+ pump under control conditions. They can be induced by brief culture in the presence of low concentrations of ouabain to express a ouabain-resistant, amiloride-sensitive Na+-K+ pumping activity. They revert to the uninduced phenotype shortly after removal of ouabain from the culture medium. When placed in low-potassium, low-serum, or dibutyryl adenosine 3'-5'-cyclic monophosphate-containing media, these cells produce long cellular extensions. Thus a single inducible gene appears to regulate sensitivity of the Na+-K+ pumping activity to cardiac glycosides and to amiloride, and it may be related to important characteristics of cellular morphology.
Transport of 3-hydroxy[3-14C]butyrate by dissociated cells from rat brain
Authors: Tildon J. T. , Roeder L. M.
Published: December 2017

Recent studies suggest that the utilization of oxidizable substrates by the brain may be regulated in part by transport across the plasma membrane. Dissociated brain cells obtained by mechanical disruption of rat brain were used to measure the uptake of 3-hydroxy[3-14C]butyrate. Total uptake revealed two mechanisms (diffusion and a carrier-mediated system). A Lineweaver-Burk plot of the latter component yielded an apparent Km of 1.47 mM and a maximal velocity (Vmax) of 5 nmol.min-1.mg protein-1. The rates of uptake were temperature dependent and were significantly higher at pH 6.2 than at pH 7.4 or 8.2. Preloading the cells and increasing the intracellular concentration of 3-hydroxybutyrate using 12.5 and 25 mM increased the rate of uptake 143 and 206%, respectively, indicative of an accelerative exchange mechanism. Uptake was inhibited approximately 50% by (in mM) 10 phenylpyruvate, 10 alpha-ketoisocaproate, 10 KCN, and 1.5 NaAsO2. Uptake was also decreased by (in mM) 5 lactate, 5 methyl malonic acid, 1 alpha-cyano-4-hydroxycinnamate, and 1 mersalyl. Dissociated brain cells from 14- to 16-day-old rats accumulated 3-hydroxybutyrate at a rate more than two-fold greater than cells from either younger (2-day-old) or older (28-day-old and adult) animals. These data are consistent with the proposal that 3-hydroxybutyrate is taken up by the brain by both diffusion and a carrier-mediated transport system, and they support the hypothesis that transport at the cellular level contributes to the regulation of substrate utilization by the brain.
G-1,6-P2 in human skeletal muscle after isometric contraction
Authors: Katz A. , Lee A. D.
Published: December 2017

The content of glucose 1,6-bisphosphate (G-1,6-P2), an in vitro activator of phosphofructokinase (a rate-limiting enzyme for glycolysis), and the glycolytic rate in skeletal muscle during isometric contraction have been determined. Subjects contracted the knee extensor muscles at two-thirds maximal voluntary force to fatigue. Biopsies from the quadriceps femoris muscle were obtained before and immediately after contraction. G-1,6-P2 increased in all subjects from a mean of 101 +/- 15 (SE) mumol/kg dry wt at rest to 128 +/- 24 at fatigue (P less than 0.05). Muscle glucose did not change significantly, whereas hexosemonophosphates were significantly increased after contraction. The glycogenolytic and glycolytic rate averaged 70.0 +/- 13.8 and 47.3 +/- 6.7 mmol.kg dry wt-1.min-1, respectively, and the glycolytic rate was positively correlated with the accumulation rates of fructose 6-phosphate (F-6-P) (r = 0.95, P less than 0.01) and G-6-P (r = 0.96, P less than 0.01). Phosphocreatine and ATP decreased by 87 and 17%, respectively, whereas ADP increased by 31% after contraction. These data demonstrate that intense, short-term isometric contraction results in an elevation of the muscle content of G-1,6-P2. The increase in G-1,6-P2 could not be accounted for by the side reactions of phosphoglucomutase or phosphofructokinase. It remains to be determined whether the observed increase in G-1,6-P2 is sufficient to account for the high glycolytic rate during intense exercise. The lack of increase in muscle glucose while G-6-P increased (which will inhibit hexokinase) suggests that the debranching enzyme complex was not active during contraction.
Mammalian blastocyst: transport functions in a developing epithelium
Authors: Biggers J. D. , Bell J. E. , Benos D. J.
Published: December 2017

This article reviews the current state of knowledge concerning morphological and physiological mechanisms important to growth and differentiation of the mammalian blastocyst between compaction and implantation. Morphological processes occur in conjunction with major changes in transport systems that control the movement of substances into and out of the embryo. Compaction is a morphological development that is associated with the formation of an outer squamous epithelium, the trophectoderm, which regulates the composition of the medium bathing the presumptive embryo (the inner cell mass). Implantation involves the interaction of two epithelia, the adhesion between the trophectoderm and the maternal endometrium. Before adhesion, the blastocyst lies free in the uterine fluid and exchanges occur between this fluid and the embryo. Apposition of these epithelia is brought about in part by expansion of the blastocyst and removal of the uterine fluid. Blastocyst physiology is an inherently important field because vectorial transport system development and the genes that regulate it can be studied.
Activation of a calcium-permeable cation channel by insulin-like growth factor II in BALB/c 3T3 cells
Authors: Matsunaga H. , Nishimoto I. , Kojima I. , Yamashita N. , Kurokawa K. , Ogata E.
Published: December 2017

Insulin-like growth factor II (IGF II) is a member of somatomedin family and is a potent mitogen in various types of mammalian cells. We have recently reported that IGF II stimulates calcium influx in competent BALB/c 3T3 cells primed with epidermal growth factor (J. Biol. Chem. 262: 12120-12126, 1987). Using patch-clamp technique, we show here an IGF II-sensitive cation channel in BALB/c 3T3 cell plasma membrane. Calcium is permeable to this cation channel and its opening behavior is independent of membrane potential. IGF II increases the opening probability of the identical channel in cells pretreated sequentially with platelet-derived growth factor and epidermal growth factor, whereas IGF II does not affect the opening of the channel in G0-arrested cells. This cation channel activity is observed only when IGF II is included in the patch pipette, indicating direct regulation of the channel by IGF II. We suggest that IGF II stimulates calcium influx by opening this cation channel and that IGF II activates the channel in a unique cell cycle-dependent manner.
Differentiated properties of rabbit tracheal epithelial cells in primary culture
Authors: Liedtke C. M.
Published: December 2017

The purpose of this study was to establish whether rabbit tracheal epithelial cells, grown in primary cell culture, retain neurohormonal receptor and mediator activity. Epithelial cells were isolated by enzymatic treatment and cultured on a collagen matrix. The culture medium consisted of a 1:1 mixture of Dulbecco's modified Eagle medium and Ham's F12 supplemented with 5% fetal calf serum, epidermal growth factor, insulin, transferrin, fibronectin, hydrocortisone, and trace elements. Cultures had a population doubling time of 48 h. Mucus-containing cells and cilia were not observed after 7 days of incubation. Positive immunofluorescent staining with monoclonal antibodies to keratins established the epithelial nature of the cell cultures. Primary cell cultures responded to beta-adrenergic agonists with a dose- and time-dependent increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Propranolol, a beta-adrenergic antagonist, blocked the effects of the beta-adrenergic agonist. Adrenergic agonists also mediated a dose-dependent increase in the generation and release of prostaglandin E2 (PGE2). PGE2 caused an increase in cAMP levels. The results demonstrate that primary cultures of rabbit tracheal epithelial cells retain hormonal responses of the isolated epithelium and tracheal mucosa-submucosa.
Identification and transmembrane signaling of leukotriene D4 receptors in human mesangial cells
Authors: Simonson M. S. , Mene P. , Dubyak G. R. , Dunn M. J.
Published: December 2017

Although peptidoleukotriene (LTC4, LTD4) receptors have been characterized by radioligand binding studies, pathways of transmembrane signaling by activated leukotriene receptors remain obscure. We employed [3H]LTD4 binding studies and fluorescent measurements of intracellular Ca2+ concentration ([Ca2+]) and pH to identify LTD4 receptors and mechanisms of transmembrane signaling in cultured human mesangial cells. Mesangial cells expressed a single class of saturable, specific binding sites for [3H]LTD4. Kinetic, competition, and saturation analyses gave an average KD of approximately 12.0 nM with a Bmax of 987 fmol/mg protein. LTC4 competed with high affinity for [3H]LTD4 binding sites, as did LTB4 but with much lower affinity. [3H]LTD4 binding was blocked by a specific LTD4 receptor antagonist, SKF 102922. LTD4 and LTC4 also evoked a rapid (2-3 s), transient increase in intracellular [Ca2+], followed by a second, sustained increase. The transient phase was independent of extracellular Ca2+, whereas the sustained phase was dependent on extracellular Ca2+. Intracellular [Ca2+] was unaffected by LTB4. The LTD4-stimulated Ca2+ transients were dose dependent (1 nM-1 microM) and, similar to [3H]LTD4 binding, Ca2+ transients were inhibited by LTD4 receptor antagonists. We also report evidence that LTD4 affects intracellular pH and activates Na+-H+ exchange. Specifically, LTD4 induced an initial acidification within 1-2 min, followed by net alkalinization at 5 min. Alkalinization was due to activation of an amiloride-inhibitable Na+-H+ exchanger. LTD4 receptors were apparently not coupled to adenylate cyclase or phospholipase A2 as we detected no changes of adenosine 3',5'-cyclic monophosphate (cAMP) or prostanoids. Thus we conclude that [3H]LTD4 binding sites on human mesangial cells are coupled to a Ca2+-signaling system and Na+-H+ exchange. Moreover LTD4, a potent inflammatory mediator, failed to stimulate cAMP or prostaglandin E2/prostaglandin I2, two counterregulatory autacoids that preserve normal mesangial function.
Response of aequorin-loaded platelets to activators of protein kinase C
Authors: Ware J. A. , Saitoh M. , Smith M. , Johnson P. C. , Salzman E. W.
Published: December 2017

The protein kinase C activators phorbol ester 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (DAG) cause platelet aggregation, secretion, and a rise in aequorin-indicated cytoplasmic Ca2+ ([Ca2+]i), but the importance of this action to platelet activation by these agonists has not been established. We found that the previous addition of PMA or DAG either enhanced or inhibited the platelet response if thrombin was subsequently added, depending on the latter's concentration. The effects of PMA or DAG on the response to thrombin were obtained only if the agonists were added in concentrations sufficient to elevate [Ca2+]i themselves. A [Ca2+]i rise also occurred after the second agonist (thrombin), but its magnitude did not necessarily correlate with subsequent aggregation, secretion, or the activation of protein kinase C as reported by the phosphorylation of a 47-kDa protein (p47). The protein kinase C inhibitor sphingosine inhibited aggregation and p47 phosphorylation caused by PMA or DAG alone or with thrombin, but the [Ca2+]i rise in response to the first agonist was not affected. PMA-induced aggregation and p47 phosphorylation were inhibited by quin2, which also inhibited protein kinase C activity in a cell-free system. We conclude that a rise in aequorin-indicated [Ca2+]i is necessary for PMA or DAG to activate platelets or to alter the subsequent platelet response to thrombin; this [Ca2+]i rise may be a prerequisite for activation of protein kinase C.
Cross-bridge dephosphorylation and relaxation of vascular smooth muscle
Authors: Hai C. M. , Murphy R. A.
Published: December 2017

We tested the hypothesis that relaxation in vascular smooth muscle is the result of inactivation of myosin light chain kinase and cross-bridge dephosphorylation. Fast neurally mediated contractions of swine carotid medial strips were induced by electrical field stimulation. Termination of the stimulus resulted in relaxation with a half time of 2 min. Nifedipine (0.1 microM) increased the relaxation rate without significant effects on the contractile response. Cross-bridge dephosphorylation was much faster than stress decay with basal levels reached within 1 min when 73% of the developed stress remained. The time-course data of dephosphorylation and stress were analyzed with a model that predicted the dependences of stress and isotonic shortening velocity on cross-bridge phosphorylation during contraction. Rate constants resolved from contraction data also fitted the relaxation data when the model's prediction was corrected for estimated errors in the phosphorylation measurements. Because Ca2+-dependent cross-bridge phosphorylation was the only postulated regulatory mechanism in the model, these results are consistent with the hypothesis that cross-bridge dephosphorylation is necessary and sufficient to explain relaxation in the swine carotid media.
Increase vs. decrease of calcium uptake by isolated heart cells induced by H2O2 vs. HOCl
Authors: Kaminishi T. , Matsuoka T. , Yanagishita T. , Kako K. J.
Published: December 2017

Adult rat heart myocytes were labeled rapidly with exogenous [45Ca2+]. Addition of 2.5 mM H2O2 to the heart cell suspension raised the content of rapidly exchangeable intracellular Ca2+ twofold, whereas addition of 1-30 mM HOCl decreased the Ca2+ content. The H2O2-induced increase in Ca2+ content was dependent on the medium Na+, pH, and temperature but was not significantly affected by addition of verapamil, diltiazem, amiloride, or 3-aminobenzamide. The [3H]ouabain binding to myocytes was suppressed by H2O2, whereas the Ca2+ efflux from myocytes was not influenced. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, reduced Ca2+ content, implying that the H2O2-induced change in Ca2+ content was not directly related to ATP depletion. On the other hand, the H2O2-induced Ca2+ accumulation in myocytes was prevented by deferoxamine or o-phenanthroline. These results suggest that H2O2 inhibited Na+-K+-ATPase, resulting in an increase in intracellular Na+ concentration and stimulation of sarcolemmal Na+-Ca2+ exchange activity, which caused a transient net Ca2+ influx into myocytes. By contrast, HOCl decreased the Ca2+ content of the rapidly exchangeable pool below control levels and this action of HOCl was antagonized by 1,4-dithiothreitol. HOCl accelerated Ca2+ efflux from myocytes. Ca2+ uptake and Ca2+-ATPase of the isolated sarcoplasmic reticular (SR) fraction were highly sensitive to the action of HOCl. Ca2+ uptake by intracellular sites, studied with myocytes permeabilized with digitonin, was inhibited by both H2O2 and HOCl. Thus these results suggest that HOCl inhibits the SR Ca2+ pump, resulting in the observed acceleration of Ca2+ efflux from and decline in Ca2+ content of myocytes.
Regulation of cAMP metabolism by PGE2 in cortical and medullary thick ascending limb of Henle's loop
Authors: Nakao A. , Allen M. L. , Sonnenburg W. K. , Smith W. L.
Published: December 2017

We have examined the regulation by prostaglandin E2 (PGE2) of hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cells isolated by immunodissection from both the medullary and cortical thick ascending limb of Henle's loop of rabbit kidney. At concentrations greater than 10(-8) M, PGE2, but not sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), caused cAMP accumulation in both cortical and medullary thick limb cells. However, at concentrations of less than or equal to 10(-8) M, both PGE2 and sulprostone inhibited arginine vasopressin (AVP)-, calcitonin-, and glucagon-induced cAMP accumulation in medullary thick ascending limb (mTAL) cells. In cortical thick limb (cTAL) cells, sulprostone also inhibited AVP-, calcitonin-, and parathyroid hormone (PTH)-induced cAMP accumulation. The inhibitory effects of PGE2 and of sulprostone were blocked by pretreatment of mTAL and cTAL cells with pertussis toxin. Membranes prepared from mTAL cells exhibited a [3H]PGE2 binding activity that was stimulated on addition of the stable guanosine 5'-triphosphate (GTP) analogue, 5'-guanosine gamma-thiotriphosphate (GTP gamma S); moreover, sulprostone inhibited [3H]PGE2 binding. Our results suggest that PGE2 can function via a prostaglandin E receptor linked to a guanine nucleotide regulatory protein, Gi, to attenuate hormone-induced cAMP formation in both mTAL and cTAL cells of rabbit kidney.
Turnover of adenosine in plasma of human and dog blood
Authors: Moser G. H. , Schrader J. , Deussen A.
Published: December 2017

To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a "stopping solution." Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added [3H]adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole.(ABSTRACT TRUNCATED AT 250 WORDS)
Influx of L-arginine is an indicator of membrane potential in human fibroblasts
Authors: Bussolati O. , Laris P. C. , Nucci F. A. , Dall'Asta V. , Franchi-Gazzola R. , Guidotti G. G. , Gazzola G. C.
Published: December 2017

The net influx of L-arginine (JARG) was employed as an indicator of the membrane potential in human fibroblasts. Cell depolarization, obtained by increasing [K+]out, decreased both JARG and the net influx of the lipid soluble cation tetraphenylphosphonium (JTPP), a probe of membrane potential. JTPP, but not JARG, was influenced by the mitochondrial potential and exhibited a component dependent on intracellular and/or extracellular binding. JARG was sensitive to changes in the membrane potential induced by Na+-dependent transport of L-proline or by the activity of Na+-K+-ATPase. In the presence of 50 microM valinomycin, JARG was markedly influenced by the distribution ratio of K+ in a range of [K+]out from 1.5 to 100 mM. In this range of [K+]out, membrane potential (Em) varied from -90 to -23 mV, and calibration of JARG vs. the membrane potential yielded a linear relationship. These results indicate the following: 1) that the net influx of TPP+ is not a reliable indicator of membrane potential in cultured human fibroblasts; 2) that in the same cells the net influx of L-arginine can be employed as an index of membrane potential; 3) that in a range of Em from -23 to -90 mV the activity of system y+ (the membrane agency devoted to L-arginine transport in cultured human fibroblasts) exhibits no saturation of potential-dependent activation of transport.
Single muscle fiber enzyme shifts with hindlimb suspension and immobilization
Authors: Fitts R. H. , Brimmer C. J. , Heywood-Cooksey A. , Timmerman R. J.
Published: December 2017

The purpose of this investigation was to determine how models of weightlessness, hindlimb suspension (HS), and hindlimb immobilization (HI) affect the metabolic enzyme profile in the slow oxidative (SO), fast oxidative glycolytic (FOG), and fast glycolytic (FG) fibers of rat hindlimb. After 1, 2, or 4 wk of HS or HI, single fibers were isolated from freeze-dried soleus and gastrocnemius muscles; a small section of each fiber was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels to identify fiber type, and the remaining piece was assayed for either lactate dehydrogenase (LDH) and citrate synthase (CS) or phosphofructokinase (PFK) and beta-hydroxyacyl-CoA dehydrogenase (beta-OH-acyl-CoA). Two weeks of HS induced an almost twofold increase in the activity of CS (2.13 +/- 0.13 vs. 3.60 +/- 0.26 mol.kg dry wt-1.h-1) in the SO fiber of the soleus, and the activity stayed high at 4 wk. Although the FOG fiber had significantly higher CS activity (3.85 +/- 0.29) than either the SO or FG (1.59 +/- 0.16 mol.kg dry wt-1.h-1) fiber, neither fast fiber type was altered by HS. The glycolytic enzymes LDH and PFK were both elevated in the SO fiber after HS. The increase in LDH occurred by 1 wk (14.80 +/- 1.51 vs. 8.83 +/- 0.78), whereas the activity of PFK was not significantly changed until 4 wk (1.16 +/- 0.13 vs. 0.68 +/- 0.05 mol.kg dry wt-1.h-1). The control FG fiber had the highest LDH (44.30 +/- 2.29) and PFK (2.40 +/- 0.16) activities, followed by the FOG fiber (LDH, 34.10 +/- 2.83; PFK, 1.62 +/- 0.17 mol.kg dry wt-1.h-1); however, the activities of these glycolytic enzymes in the fast fiber types were unaltered by HS. The activity of beta-OH-acyl-CoA was not affected by HS in either the slow or fast fiber types. HI showed qualitatively similar changes to those observed with HS; however, the enzyme shifts developed with a slower time course. In conclusion, both HS and HI shifted the SO fiber enzyme pattern toward that of the control FOG fiber; however, a complete conversion from the SO to FOG fiber did not occur within the 4-wk treatment period.
Alterations of cytosolic calcium in LLC-PK1 cells induced by vasopressin and exogenous purines
Authors: Weinberg J. M. , Davis J. A. , Shayman J. A. , Knight P. R.
Published: December 2017

The regulation of cytosolic calcium in LLC-PK1 cells by various agonists was characterized. Arginine vasopressin (AVP, 100 nM) rapidly increased cytosolic calcium (Caf) measured with fura-2 from a basal level of 65 +/- 5 to 516 +/- 102 nM followed by a return to a plateau level of 128 +/- 18 nM. Similar responses to 100 nM lysine vasopressin were seen. AVP also increased adenosine 3',5'-cyclic monophosphate (cAMP) as previously documented for these cells. A V2-selective AVP analogue increased cAMP without affecting Caf, whereas two V1-receptor antagonists prevented the Caf response to AVP without altering the cAMP response. Increasing cellular cAMP with forskolin, cholera toxin, or stable cAMP analogues did not affect Caf or the response of Caf to AVP. Both adenosine and ATP produced large Caf transients at concentrations of 1-10 microM in both calcium-containing media and after acute chelation of medium Ca with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The A1-selective adenosine analogue, (R-phenyl-isopropyl)-adenosine, and the A2-selective analogue, 5'-(N-ethyl)-carboxamido-adenosine, both produced Caf responses similar to adenosine. The Caf responses to adenosine and its analogues but not to ATP were blocked by the adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Islet-activating protein, pertussis toxin, inhibited the Caf response to adenosine and enhanced the cAMP response to AVP. Responses to all agonists were demonstrable in greater than 80% of single cells studied by microfluorometry, and individual cells responded to multiple agonists. These studies indicate that the Caf and cAMP responses to AVP in the LLC-PK1 cell line involve separate receptors, and they document the presence in this cell line of at least two types of receptors for exogenous purines.
Metabolic fiber types of snake transversus abdominis muscle
Authors: Wilkinson R. S. , Nemeth P. M.
Published: December 2017

Fibers of the garter snake transversus abdominis muscle fall into three classes according to contraction speed: faster and slower twitch and tonic. To determine the relationship between these physiologically determined classes and established mammalian fiber types, individual fibers were assayed for key enzymes representing the major energy-generating pathways in vertebrate muscle. Five such enzymes were examined: lactate dehydrogenase, malate dehydrogenase, adenylokinase, fumarate hydratase, and beta-hydroxyacyl-CoA dehydrogenase. The muscle contained three principal metabolic fiber types. Fast-contracting twitch fibers had low-oxidative but high-glycolytic capacity and therefore resembled mammalian-type fast-twitch glycolytic (FG) fibers. Slower twitch fibers were high oxidative-high glycolytic, similar to mammalian-type fast-twitch, oxidative, glycolytic (FOG) fibers. Tonic fibers were high oxidative-low glycolytic; this metabolic profile is characteristic of type slow-twitch oxidative (SO) fibers in mammals. Activity of the enzyme adenylokinase, which in mammals correlates with contraction speed and myosin adenosine triphosphatase (ATPase) activity, separated these reptilian fibers into three groups that are similar but not identical to those delineated by oxidative and glycolytic enzymes. Adenylokinase and beta-hydroxyacyl-CoA dehydrogenase showed the widest range of activities in snake muscle and, therefore, the greatest ability to discriminate fiber types.
Correction of proton and Ca association constants of EGTA for temperature and ionic strength
Authors: Harrison S. M. , Bers D. M.
Published: December 2017

The association constants of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for protons and Ca can be used to calculate the apparent association constant of EGTA for Ca (K'CaEGTA). This value is often used in calculating the free [Ca2+] of complex solutions such as those used to determine the Ca sensitivity of skinned muscle fibers. As association constants are usually measured at 0.1 M ionic strength and between 20 and 25 degrees C, these constants must first be adjusted for conditions different from those at which they were measured, before calculation of K'CaEGTA. The proton and Ca association constants (and their delta H values) from A. E. Martell and R. M. Smith (Critical Stability Constants, New York: Plenum, vol. 1, 1974) adjusted for ionic strength and temperature using a semiempirical Debye-Huckel formalism and Van't Hoff isochore, respectively, closely describe the influence of both ionic strength and temperature on K'CaEGTA. Errors in the adjustment or calculation of association constants can dramatically affect the calculated value of K'CaEGTA and hence the calculated free [Ca2+] of complex solutions, such as those used to mimic the intracellular environment.
Reduction in sensitivity to Cl- channel blockers by HCO3- -CO2 in rabbit cortical collecting duct
Authors: Matsuzaki K. , Schuster V. L. , Stokes J. B.
Published: December 2017

We examined the ability of HCO3- -CO2 to modify the potency of Cl- channel blockers in the renal cortical collecting duct (CCD) for the following two reasons. 1) From a practical point of view, there is, to our knowledge, no information regarding the effect of the HCO3- -CO2 buffer system on the potency of Cl- channel blockers. 2) We showed in the companion manuscript [Am. J. Physiol. 257 (Cell Physiol. 26): C94-C101, 1989] that HCO3- -CO2 stimulates transepithelial anion exchange in the CCD. Based on precedent in the literature, we postulated that HCO3- stimulates the basolateral membrane Cl- conductance. Here, we demonstrate that several Cl- channel blockers can reduce CCD Cl- self exchange when the solutions are buffered in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Concentrations of blockers producing 80% inhibition in HEPES, pH 7.4, produced only 20% inhibition in 25 mM HCO3- -CO2, pH 7.4. The ability of HCO3- -CO2 to reduce blocker potency had an IC50 of only 2 mM. We also examined interactions of HCO3- -CO2 and blockers with regard to the principal cell basolateral Cl- conductance. Blockers did not alter the Rb+ flux, a marker of K+ transport, but did reduce transepithelial conductance (GT), i.e., the blockers inhibited the principal cell basolateral Cl- conductance. As was the case with intercalated cell anion exchange, GT measurements indicated that HCO3- -CO2 impaired the ability of Cl- channel blockers to inhibit the principal cell Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)
Transport and metabolism of ascorbic acid in human placenta
Authors: Choi J. L. , Rose R. C.
Published: December 2017

The role of human placenta in cellular transport and metabolism of the potentially toxic oxidized form and the useful reduced form of ascorbic acid was examined in surviving tissue fragments in vitro. At the end of a 60-min incubation with the 14C label nominally present in the reduced form, a tissue-to-medium ratio in excess of unity was reached. The importance of evaluating uptake of the ascorbic acid metabolites is evident from a careful assay of 14C label present in the bathing media. Significant spontaneous oxidation occurs, which is slowed or reversed to a limited extent by the presence of placental tissue. Uptake of the oxidized substrate, dehydro-L-ascorbic acid, proceeds much more rapidly than uptake of ascorbic acid. At the end of a 15-min incubation, most of the substrate taken up was in the reduced form. From an additional evaluation of 14C label in the bath it is calculated that 25% of ascorbic acid formed by the tissue is released within 15 min. The cellular uptake mechanism for dehydro-L-ascorbic acid is not shared by glucose and is not dependent on the presence of Na+ but is dependent on intact cellular metabolism. The finding of avid cellular uptake and reduction of the oxidized form of ascorbic acid supports the concept that the placenta helps to clear the toxic molecule from the maternal circulation, metabolizes it, and delivers the useful reduced form to the fetus.
Histamine concentration and Ca2+ mobilization in arterial smooth muscle
Authors: Rembold C. M. , Murphy R. A.
Published: December 2017

Smooth muscle contraction is dependent on Ca2+ entry from the extracellular space or release from intracellular stores. The sensitivity of these Ca2+ sources to agonist concentration was evaluated by measuring myoplasmic [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in the swine carotid media. High histamine concentrations produced transient elevations in [Ca2+] and phosphorylation with rapid generation of near maximal stress. Lower histamine concentrations produced much smaller [Ca2+] and phosphorylation transients, and stress development was slower. Peak [Ca2+] was proportional to the rate of stress development. Steady-state [Ca2+], phosphorylation, and stress values (which are dependent on extracellular Ca2+) were more sensitive to histamine concentration than was the peak [Ca2+] response both in the presence and absence of extracellular CaCl2 (measures of intracellular Ca2+ release). This result suggests that the mechanism for Ca2+ influx from the extracellular space is more sensitive to histamine than intracellular Ca2+ release. These results are also consistent with the hypothesis that agonist-releasable sarcoplasmic reticular Ca2+ is the major contributor to initial phosphorylation transients that enhance the rate of stress development.
Caffeine-induced Ca2+ release activates Ca2+ extrusion via Na+-Ca2+ exchanger in cardiac myocytes
Authors: Callewaert G. , Cleemann L. , Morad M.
Published: December 2017

Rapid application of caffeine in fura-2-dialyzed and whole cell-clamped rat and guinea pig ventricular myocytes activated reversibly large intracellular Ca2+ transients that accompanied Na+-dependent transient inward currents. Such transient inward currents had the same time course as the intracellular Ca2+ transient and were suppressed by Ni2+ and removal of extracellular Na+. Because Ca2+ release signals were not altered by addition of Ni2+ or removal of Na+, we concluded that the rise in intracellular Ca2+ concentration was necessary for the activation of the transient inward current. Thus the caffeine-induced transient inward current represents efflux of Ca2+ via the Na+-Ca2+ exchanger.
Na+-Ca2+ exchange in bovine rod outer segments requires and transports K+
Authors: Schnetkamp P. P. , Basu D. K. , Szerencsei R. T.
Published: December 2017

Intact outer segments isolated from bovine retinas (bovine ROS) display a high activity of Na+-Ca2+ exchange, and Na+-Ca2+ exchange appears to be the only functional ion transporter present. Here we demonstrate for the first time that Na+-Ca2+ exchange requires and transports K+ from the following observations. 1) Na+-Ca2+ exchange in bovine ROS required the simultaneous presence of K+ and Ca2+ on one side of the membrane and the presence of Na+ on the other side. 2) Na+-stimulated Ca2+ release from bovine ROS was accompanied by an equally large release of K+. We used the electrogenic protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) as an added electrical shunt; in the intact rod cell, electrogenic Na+-Ca2+ exchange is shunted by K+ channels present in the rod inner segment. In the presence of FCCP, an inward Na+-Ca2+ exchange current was accompanied by an outward current of protons with a stoichiometry of 1 H+/Ca2+; in the absence of FCCP, no Na+-induced proton current was observed. Addition of FCCP did not uncouple Na+-induced K+ release from Na+-induced Ca2+ release. We conclude that Na+-Ca2+ exchange in bovine rod photoreceptors operates at an electrogenic stoichiometry of 4 Na+:(1 Ca2+ + 1 K+). In isolated ROS and in the absence of an external electrical shunt, Na+-Ca2+ exchange operated at an electroneutral stoichiometry of 3 Na+:(1 Ca2+ + 1 K+).
Phenylarsine oxide inhibition of endocytosis: effects on asialofetuin internalization
Authors: Gibson A. E. , Noel R. J. , Herlihy J. T. , Ward W. F.
Published: December 2017

The aim of this work was to establish to what extent the concentrations over which phenylarsine oxide (PAO) inhibits oxygen consumption and decreases cellular ATP content overlap with those used to inhibit protein internalization. The effects of PAO (1-100 microM) on 125I-labeled asialofetuin internalization, oxygen consumption, ATP content, and lactate dehydrogenase (LDH) latency of isolated rat hepatocytes were determined. Ten micromoles/liter PAO blocked 125I-asialofetuin internalization but had no effect on ATP content up to 20 min, only a slight inhibition (18%) on oxygen consumption, and no effect on LDH latency. Higher concentrations of PAO had increasingly deleterious effects on the parameters. These results show that higher concentrations of PAO severely affect the energy stores as well as integrity of the hepatocyte. We concluded that 1) use of PAO requires careful evaluation of its effects in each experimental preparation and 2) it should be possible to establish time and concentration parameters for use of PAO as an inhibitor of endocytosis while minimizing the influence of its other cellular actions.
Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes
Authors: Janas T. , Bjerrum P. J. , Brahm J. , Wieth J. O.
Published: December 2017

The capnophorin (band 3)-mediated chloride self exchange flux in intact erythrocytes and in resealed erythrocyte ghosts was determined at pH 7.3 by measuring the unidirectional efflux of 36Cl-. The time-dependent irreversible inactivation of the anion transport system by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was measured as the relative change of the unidirectional 36Cl efflux rate. The rate of covalent DIDS binding under conditions of excess DIDS in solution that ensure a complete irreversible inhibition followed an exponential time course with a rate coefficient Kcov (min-1). The Arrhenius activation enthalpy of Kcov was constant, 114 kJ/mol, at 0-38 degrees C. At 38 and 0 degrees C, Kcov was 0.5 min-1 [half time (T1/2) = ln2/Kcov = 1.3 min] and 0.004 min-1 (T1/2 = 178 min), respectively. The slow irreversible DIDS binding to the anion transport system at 0 degrees C allows a determination of the kinetics of the reversible DIDS reaction. The pseudo first-order rate constant for binding, kon, was 3.5 X 10(5) (M.s)-1. The apparent dissociation constant, KD, determined from the steady-state binding to the erythrocyte membrane was 3.1 X 10(-8) M at an equal internal and external Cl- concentration of 165 mM (0 degrees C). The value of KD shows that DIDS is the most efficient reversible inhibitor among the stilbene derivatives so far studied. Maximum reversible inhibition by DIDS was obtained by binding of a minimum of approximately 10(6) molecules/cell membrane. The number is similar to that obtained from studies of irreversible DIDS binding.
Inhibition of angiotensinogen production by angiotensin II analogues in human hepatoma cell line
Authors: Coezy E. , Darby I. , Mizrahi J. , Cantau B. , Donnadieu M. H. , Nussberger J. , Escher E. , Chapnick B. , Corvol P.
Published: December 2017

The aim of this study was to examine in Hep G2, a human hepatoma-derived cell line, the presence of angiotensin II (ANG II) receptors and the effect of ANG II and its analogues on angiotensinogen production. The presence of ANG II receptors was demonstrated using a long-acting ANG II analogue, 125I-labeled [Sar1]ANG II. A single class of specific binding sites was identified in these cells with a dissociation constant (Kd) of 2 nM. The number and affinity of these binding sites were not changed by [Sar1]ANG II treatment over 24 h. ANG II showed an inhibitory effect on angiotensinogen production. [Sar1]ANG II also exhibited a similar inhibitory effect as that of ANG II but to a greater extent and therefore was used throughout these studies. [Sar1]ANG II inhibited angiotensinogen production in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) of 2 nM. Other ANG II analogues showed similar effects on angiotensinogen production. In order of decreasing ability, they were [Sar1]ANG II greater than [Sar1-Ala8]ANG II greater than [Sar1-Val8]ANG II greater than [Sar1-Val5-(Br5)-Phe8]ANG II greater than [Sar1-Val5-DPhe8]ANG II. Results of these studies show that the Hep G2 cell possesses specific ANG II receptors and that [Sar1]ANG II induces a dose-dependent inhibition of angiotensinogen production in this system.
Evidence for two distinct chloride uniport pathways in brown adipose tissue mitochondria
Authors: Jezek P. , Beavis A. D. , Diresta D. J. , Cousino R. N. , Garlid K. D.
Published: December 2017

Chloride permeability of the inner membrane of brown adipose tissue mitochondria was analyzed by monitoring mitochondrial swelling in KCl salts in the presence of K+ ionophores. The results indicate that the high anion conductance observed in these mitochondria is due to the presence of two separate pathways: 1) a Cl-conducting pathway that is inhibited by guanosine 5'-diphosphate (GDP) but neither by N,N'-dicyclohexylcarbodiimide (DCCD) nor by amphiphilic amines and that is found uniquely in brown adipose tissue mitochondria and 2) an inner membrane anion uniport channel that is inhibited both by DCCD and by amphiphilic amines but not by GDP and that is opened either by depletion of matrix Mg2+ or by alkalinization of the matrix.