bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionic currents in vertebrate myelinated nerve at hyperbaric pressure
Authors: Kendig J. J.
Published: December 2017

To establish a base line for a study of anesthetic-pressure antagonism in axons, voltage-clamped nodes of Ranvier from amphibian sciatic nerve were subjected to pressures of 1-100 atm. Over the time of compression, there was usually an irreversible decrease in peak inward sodium current, but there was no change in peak outward sodium current or in the current-voltage relationship. The steady-state inactivation-voltage curve was shifted 5-15 mV in the depolarizing direction at 70-100 atm. The rate of rise of the sodium current was slowed, as was the time constant of inactivation (tau h). Increase in tau h was markedly voltage dependent, suggesting a selective effect of pressure on beta h, the rate constant governing development of the inactive state. The rate of development of steady-state outward potassium current was also decreased, without significant change in maximum current. The effects of pressure are qualitatively similar to, but different in detail from, those reported in squid axon and different in some details from the effects of cooling in this preparation. None of the effects can presently be related to the high-pressure nervous syndrome.
Internal perfusion studies demonstrating GABA-induced chloride responses in frog primary afferent neurons
Authors: Hattori K. , Akaike N. , Oomura Y. , Kuraoka S.
Published: December 2017

Cl- current in gamma-aminobutyric acid (GABA)-sensitive neurons of the frog dorsal root ganglia was separated from other ionic components (i.e., Na+, Ca2+, and K+ currents) using a suction pipette technique, which allowed internal perfusion and current clamp. The GABA-induced depolarization response increased slightly on substituting Na+ by tris(hydroxymethyl)amino-methane+ or Ca2+ by Mg2+ in the external solution. Additional replacement of external and internal K+ with Cs+ further enhanced the GABA response. The GABA response was virtually unaffected when the internal perfusate contained, in addition to 60 mM Cl-, large organic anions such as isethionate-, aspartate-, and citrate- (each 70 mM) as well as aspartate- and GABA (each 35 mM). The reversal potential of GABA-induced Cl- response (EGABA) was equal to Cl- equilibrium potential (ECl) and behaved as a simple Cl- electrode following changes of external and internal Cl- concentrations. These observations indicate the adequacy of internal perfusion. The GABA-induced Cl- response increased in a sigmoidal dose-dependent manner, in which the threshold GABA concentration was around 10(-7) M and the GABA response reached ECl at 10(-4) M. When GABA concentration was higher than 6 X 10(-6) M, the responses were always accompanied by a rapid desensitization.
Effect of calmodulin, Ca2+, and cAMP protein kinase on skinned tracheal smooth muscle
Authors: Sparrow M. P. , Pfitzer G. , Gagelmann M. , Ruegg J. C.
Published: December 2017

The characteristics of contraction and relaxation of membrane skinned smooth muscle from guinea pig trachealis muscle are described. Micromolar Ca2+ elicited reproducible contractions in Mg-ATP salt solution at 20 degrees C. The speed of contraction was much faster at 30 and 37 degrees C, enabling cumulative concentration-response curves to be obtained. At these temperatures, a progressive increase in basal tension occurred in the absence of Ca2+. This tension was active and developed more rapidly at pH 6.7 than at pH 7.0. Calmodulin (0.1-10 microM) greatly increased the speed of contraction and lowered the threshold Ca2+ concentration ([Ca2+]) required to initiate contraction from 0.13 to 0.02 microM Ca2+. Trifluoperazine antagonized responses to Ca2+. Thiophosphorylation with adenosine 5'-O-(3-thiotriphosphate) produced maximum tension development, which was Ca2+-independent. This effect was reversible. The results are compatible with myosin-linked regulation of contraction in which a Ca2+ X calmodulin complex activates myosin light chain kinase to phosphorylate myosin. The catalytic subunit of cAMP-dependent protein kinase strongly inhibited tension development and slowly relaxed fibers contracted with threshold [Ca2+] consistent with an action via phosphorylation of myosin light chain kinase. This effect was extremely slow compared with the rate of relaxation by Ca2+ withdrawal or with relaxation of intact smooth muscle by beta-adrenergic agonists.
Stretch-induced growth in chicken wing muscles: nerve-muscle interaction in muscular dystrophy
Authors: Ashmore C. R. , Lee Y. B. , Summers P. , Hitchcock L.
Published: December 2017

Skeletal muscle growth following denervation and denervation plus passive stretch was characterized in the patagialis muscle of normal and dystrophic chicks until 8 wk of age. In both genotypes, muscles denervated at 1 wk of age grew at reduced rates compared with contralateral control muscles whether or not they were passively stretched. Histograms of fiber size distributions as well as morphological criteria showed that passive stretch of denervated dystropic muscles substantially delayed the development of pathology. Denervation alone provided less protection. There was no evidence of fiber necrosis in any denervated dystrophic muscle, although many fibers did exhibit extreme hypertrophy and abnormal morphology. When denervated dystrophic muscles were allowed to reinnervate, growth and development of pathology was rapid. Because denervation, denervation with passive stretch, or passive stretch alone retards, but does not prevent, the development of pathology, it is concluded that dystrophy in the chick is a myogenic defect that is exacerbated by neurally mediated contractile activity.
Extracellular calcium ion depletion induces release of glycoproteins by canine trachea
Authors: Coles S. J. , Bhaskar K. R. , O'Sullivan D. D. , Reid L. M.
Published: December 2017

The effect of depletion of extracellular Ca2+ on the release by canine trachea in vitro of [14C]glucosamine-labeled trichloroacetic-phosphotungstic acid-precipitable glycoproteins was evaluated. Incubation in Ca2+,Mg2+-free medium containing ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA, 10(-3) to 10(-5) M) induced a time-dependent increase in base-line release of high molecular weight, radiolabeled glycoprotein that was not blocked by 10(-5) M atropine, propranolol, or phentolamine. Maximal increase was proportional to EGTA concentration and was augmented by preincubation with methacholine chloride. The secretory response was reversed by reincubation of explants in medium containing Ca2+, Sr2+, or Ba2+ but not Mg2+. Assessment of explants denuded of epithelium or cartilage indicated that the secretory response to depletion of Ca2+ originated in the tracheal submucosa, probably the submucosal glands. Quantitative autoradiographic analyses showed, however, that incubation in Ca2+,Mg2+-free medium had no effect on radiolabel release from mucous or serous cells in the tracheal submucosal glands. Increased radiolabeled glycoprotein release in Ca2+,Mg2+-free medium was accompanied by exfoliation of the surface epithelium, though the two effects were apparently unrelated. The secretory response was not due to cell lysis or increased release of radiolabeled glycosaminoglycans, and the finding that the specific activity (ratio of bound radiolabel to protein content) of the glycoprotein released was not changed in Ca2+,Mg2+-free medium showed that it was not due to a change in the rate of glycoprotein synthesis. A model is proposed in which depletion of Ca2+ increases the rate of flow of mucus from the duct lumens of the tracheal glands.
Chloride transport properties of human leukemic cell lines K562 and HL60
Authors: Dissing S. , Hoffman R. , Murnane M. J. , Hoffman J. F.
Published: December 2017

The Cl- transport characteristics of the human leukemic cell lines K562 and HL60, with erythroid and granulocytic phenotypic features, respectively, were investigated. Cl- effluxes were measured with 36Cl- under equilibrium conditions in both cell lines and were found to be three orders of magnitude slower than the unidirectional efflux of Cl- in normal erythrocytes. Induction of differentiation of the K562 cell line with hemin does not affect the rate of Cl- transport, while induction of the HL60 cell line with dimethyl sulfoxide results in a small decrease in the rate of Cl- transport. Cl- transport in both cell lines could be divided into two components. One component is inhibited by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), displays counter-transport characteristics, and has a high energy of activation--all properties characteristic of the human erythrocyte-facilitated anion exchange system. The second component is insensitive to DIDS, is partially inhibited by furosemide, and has a low energy of activation.
Hormonal influence on endothelial cell angiotensin-converting enzyme activity
Authors: Krulewitz A. H. , Baur W. E. , Fanburg B. L.
Published: December 2017

The influence of various hormones on angiotensin-converting enzyme (ACE) production and release by bovine endothelial cells in culture was studied. Dexamethasone, thyroxine (T4), and triiodothyronine (T3) stimulated ACE activity in cells and their culture supernatants without affecting cell number or protein content. The stimulating effects of dexamethasone and thyroid hormones were additive, suggesting that these hormones may have different sites of action. In addition, their stimulating effects were blocked by cycloheximide, indicating that increased enzymatic activity occurred through new protein synthesis. The exposure of cells to insulin reduced ACE activity of cells and their culture supernatants without influencing cell counts or protein content; insulin also partially inhibited the stimulation of ACE activity by dexamethasone or T3. Our studies suggest that the production of ACE by endothelial cells is under hormonal regulation. Release of ACE activity into culture supernatants parallels changes in cellular ACE activity. The results supplement previous observations made in vitro and in vivo.
Endocytic uptake, transport, and catabolism of proteins by epithelial cells
Authors: Wall D. A. , Maack T.
Published: December 2017

Adsorptive and/or receptor-mediated endocytosis of proteins is a universal cell property, which is highly expressed in epithelial cells. Some absorbed proteins are transported intact across cells and in this manner subserve specialized functions such as the transference of immunity from mother to child. Mainly, however, absorbed proteins are transported to lysosomes, where they undergo complete hydrolysis to amino acids. This process is essential for the homeostasis of circulating proteins. This brief review considers the intracellular pathways taken by endocytosed proteins and the quantitative aspects and determinants of protein uptake and catabolism by epithelial cells. The topics to be briefly discussed are initial internalization sites, transport organelles (endosomes), and lysosomal and nonlysosomal pathways of transport; intracellular sorting of internalized proteins, membranes, and receptors; kinetics and selectivity of renal cell uptake of low-molecular-weight proteins and proteohormones; receptor-mediated endocytosis of larger proteins (e.g., glycoproteins) by hepatocytes; and lysosomal catabolism of absorbed proteins and its dependence on protein load and endosomal-lysosomal pH and function. The perspectives of the field and some of the outstanding unsolved problems are briefly discussed.
31P-nuclear magnetic resonance analysis of perfused single frog skins
Authors: Lin L. E. , Shporer M. , Civan M. M.
Published: December 2017

Perfusion of single frog skins has produced stability of the 31P-nuclear magnetic resonance (NMR) spectra over periods as long as 8 h at room temperature. With this approach, relatively large phosphocreatine (PCr) signals were recorded for each of the 12 frog skins studied. The ratio of the concentration of PCr to ATP was estimated to be 0.76 +/- 0.07, a value an order of magnitude larger than that previously reported. Comparison of the ratio of the intracellular concentrations of inorganic phosphate (Pi) to ATP determined in the present NMR study with that previously estimated by chemical analysis suggests that little intracellular Pi is likely to be immobilized. Inclusion of methylphosphonate in the perfusing solutions permitted simultaneous determination of extracellular and intracellular pH from the NMR spectra alone. The methylphosphonate has been found to be nontoxic. At an extracellular pH of 7.72 +/- 0.04, the intracellular pH was 7.47 +/- 0.06.
Influence of acidosis on AMP deaminase activity in contracting fast-twitch muscle
Authors: Dudley G. A. , Terjung R. L.
Published: December 2017

The rate of AMP deamination to IMP and NH4, by the action of AMP deaminase, is increased in vitro by acidosis and elevations in [AMP] and [ADP]. We evaluated the influence of acidosis on the activity of AMP deaminase in contracting muscle (5 Hz) by relating the time course of IMP and NH4 production to lactate-induced acidosis in low-oxidative, fast-twitch white (FTW) and high-oxidative, fast-twitch red (FTR) muscle of the rat. Cellular acidosis was modified by controlling lactic acid accumulation by regulating muscle blood flow and using trained animals. A significant activation of AMP deaminase occurred in both muscle types, but only at times when the estimated pH was 6.6 and below (lactate content 20 mu mol/g and above). Cellular acidosis, however, is not absolutely essential, since iodoacetic acid-blocked muscle lost 85-90% of its ATP to IMP during contractions. Thus cellular acidosis seems to be an important, but not the sole, factor activating AMP deaminase during contractions. Further, the influence of acidosis is probably different between fiber types, since the estimated free AMP and ADP contents, calculated from the creatine kinase and myokinase reactions, were different in the two fiber types. Most of the activation of AMP deaminase in FTR muscle could be attributed to a substrate effect of the increased free AMP content. In contrast, most of the activation of AMP deaminase in the FTW muscle was due to factors other than a substrate effect. These results suggest that cellular acidosis during intense contraction conditions is a major factor activating AMP deaminase, especially in the low-oxidative FTW muscle fiber type.
Cytoplasmic and nuclear uptake of aldosterone in toad bladder: a mathematical modeling approach
Authors: Claire M. , Steimer J. L. , Oblin M. E. , Gaeggeler H. P. , Venot A. , Corvol P. , Rossier B. C.
Published: December 2017

The mechanism of aldosterone uptake in the epithelial cells of toad bladder was studied using mathematical modeling. Two complementary approaches were used. The first involved analysis of cytosolic aldosterone binding at steady state according to models defined by the sum of independent noninteractive binding sites. The best model describing the experimental data corresponded to two specific binding sites with mean dissociation constant values of 0.20 and 60 nM for types 1 and 2, respectively. The second approach was based on the analysis of cytoplasmic and nuclear aldosterone uptake kinetics at 25 and 0 degrees C in intact bladder. Two models (A and B) were studied. They both implied the existence of two types of aldosterone binding sites as precursors of the corresponding chromatin bound complexes. In model A, nuclear translocation of the two types of receptors was assumed to obey first-order kinetics. In model B, the translocation process for type 1 sites involved a time lag leading to delayed binding to chromatin. Both models were found to fit the experimental data satisfactorily. The fit obtained for model B appeared to be better at low aldosterone concentrations.
Hormonal control of gap junctions in the myometrium
Authors: MacKenzie L. W. , Garfield R. E.
Published: December 2017

Myometrial tissues from immature, ovariectomized mature, and pregnant rats were examined by thin-section or freeze-fracture electron microscopy for the presence of gap junctions following treatment with steroid hormones and inhibitors of prostaglandin synthesis. Gap junctions were present in the myometrium of immature rats after repeated injections of estradiol or diethylstilbestrol (500 micrograms/day). The frequency of gap junctions in immature rats after estradiol treatment was similar to that found during delivery. Concomitant administration of indomethacin or meclofenamate with estradiol (50 and 500 micrograms/day) for 3 or 4 days enhanced the effect of estradiol on gap junctions. Progesterone depressed the response of 500 micrograms/day estradiol and both 50 and 500 micrograms/day doses of estradiol when administered together with the inhibitors of prostaglandin synthesis. Gap junctions were also present in the myometria of ovariectomized mature rats after treatment with estradiol by itself or together with indomethacin, and these effects were suppressed by concomitant progesterone administration. These results indicate that the steroid hormones and prostaglandins may interact to modulate the presence of gap junctions in the myometrium.
Effect of insulin on intracellular pH in frog skeletal muscle fibers
Authors: Putnam R. W.
Published: December 2017

The effect of insulin on intracellular pH (pHi) and membrane potential was studied in frog semitendinosus muscle fibers, using recessed-tip pH-sensitive glass microelectrodes and conventional 3 M KCl-filled microelectrodes. After a lag period of approximately 20 min, insulin [1 mU/ml, 0.1% bovine serum albumin (BSA)] produced a slow hyperpolarization of 2–5 mV and an alkalinization of 0.05–0.10 pH unit, which were both completed within 1 h and were not reversed by washing in insulin-free solution for 1 h. The effect of insulin on the pHi recovery rate from CO2-induced acidification was examined at various membrane voltages. At normal membrane voltage, insulin (400 mU/ml, no BSA) slightly increased the slow pHi recovery (from 0.01 to 0.04 delta pH/h). In fibers depolarized in 15 mM K to about -50 mV, insulin nearly tripled the recovery rate (from 0.05 to 0.13 delta pH/h). This insulin-induced recovery was abolished by 1 mM amiloride, a Na-H exchange inhibitor. The increased pHi recovery in 15 mM K thus represents an increased Na-H exchange, which may be due to an interaction between insulin and either membrane depolarization, per se, or increased intracellular Ca. In fibers depolarized in 50 mM K to about -25 mV, insulin did not affect recovery (0.28 delta pH/h). This lack of insulin effect might be due to fiber swelling or to the difference in the time course of elevation of intracellular Ca at -25 and -50 mV. These results are consistent with an alkalinizing effect of insulin in frog muscle mediated by Na-H exchange.
[3H]GDP binding and thermogenin amount in brown adipose tissue mitochondria from cold-exposed rats
Authors: Nedergaard J. , Cannon B.
Published: December 2017

Brown fat mitochondria were isolated from cold-exposed and control rats, and their content of the brown-fat-specific 32-kDa “uncoupling” protein thermogenin determined both by the traditional [3H]GDP-binding method and by the recently developed enzyme-linked immunosorbent assay (ELISA). In mitochondria isolated from both cold-acclimated (3 wk at 4 degrees C) and cold-exposed rats (24 h), an increase in thermogenin content was observable, both when estimated by the [3H]GDP-binding method and by the ELISA assay, and there was no statistically significant difference in the magnitude of these increases in the two methods. In 1 h cold-exposed rats there was no increase in [3H]GDP binding or in the ELISA reaction. When the amount of thermogenin was plotted against [3H]GDP binding in the different states, a relationship of 75,000 g thermogenin per mole GDP bound was obtained. Based on the resolution of these two methods, and under the three conditions investigated, it was concluded that there was no reason to postulate the existence of a “masked” form of thermogenin or of an “unmasking” process and that thermogenin in the mitochondria, as in the isolated state, has apparently one GDP binding site per dimer.
Effect of catecholamines on glucose uptake and glycogenolysis in rat skeletal muscle
Authors: Young D. A. , Wallberg-Henriksson H. , Cranshaw J. , Chen M. , Holloszy J. O.
Published: December 2017

The effect of catecholamines on glycogenolysis and sugar transport was evaluated in rat epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles in vitro. When muscles were incubated with 0.1 microM epinephrine (both an alpha- and beta-agonist), the proportion of phosphorylase in the a form increased from 6.2 +/- 0.7 to 37.4 +/- 5.7% in epitrochlearis muscle and from 9.1 +/- 0.7 to 21.6 +/- 1.3% in soleus muscle. Both the activation of phosphorylase and the resulting glycogenolysis could be prevented by preincubation with the beta-blocker, propranolol. The effect of catecholamines on the rate of sugar transport was also examined in epitrochlearis muscle. The beta-agonist, isoproterenol, significantly depressed the rate of 3-O-methylglucose uptake, while the alpha-agonist, phenylephrine, had no effect. Inclusion of 0.1% albumin in the incubation medium increased the resting rate of sugar transport twofold. When isoproterenol + albumin were present, rather than exerting a depressive effect the catecholamine further increased the rate of sugar uptake. This increase was prevented by preincubation with propranolol. It was concluded that glycogenolysis and sugar transport in rat skeletal muscle are solely under beta-adrenergic control.
Regulation of hormonal responsiveness in LLC-PK1L cells grown in defined medium
Authors: Roy C.
Published: December 2017

LLC-PK1L cells, a kidney-derived cell line, were able to grow in a chemically defined medium. Growth of the cells in the presence of retinol, ergocalciferol, d-alpha-tocopherol, 3,3',5-triiodothyronine, hydrocortisone, l-carnitine, d-l-methionine-S-methylsulfonium chloride, insulin, transferrin, cholesterol, and sodium linoleate increased the number of vasopressin receptors by 20- to 40-fold. All the newly detectable vasopressin receptors were coupled to the adenylate cyclase activity with similar efficiency. The same growth conditions did not alter the basal adenylate cyclase activity or the responses to calcitonin, parathyroid hormone, prostaglandin, adenosine, and GTP. In contrast, the increased responsiveness of the adenylate cyclase to vasopressin was associated with a reduced response to isoproterenol. Such an inverse correlation was also found when the time course of vasopressin receptor induction was studied. The supplemented medium permitted the growth of cells for several weeks. The effects of the enriched medium were fully reversible when we returned to the original cell growth medium. Thus such a cellular system appears as a useful tool for further work in cellular and kidney endocrinology and for detailing the molecular mechanisms of receptor-adenylate cyclase regulations.
Baboon erythrocyte ghosts contain beta-adrenergic receptors
Authors: Susanni E. E. , Ross F. P. , Scriven D. R. , Rosendorff C.
Published: December 2017

We have used the beta-adrenergic antagonist [3H]dihydroalprenolol [( 3H]DHA) to identify binding sites on the erythrocyte membrane of the primate Papio ursinus. Analysis of the saturation isotherm revealed binding to be saturable with a maximal number of binding sites of 499 fmol/mg protein. [3H]DHA binds specifically to the erythrocyte ghosts with an apparent dissociation constant (Kd) of 0.57 +/- 0.06 nM. A similar value for Kd (0.46 +/- 0.07 nM) was evaluated from the rate constants of association (0.013 +/- 0.003 X nM-1 X min-1) and dissociation (0.006 +/- 0.001 X min-1). beta-adrenergic agonists compete for the binding sites with an order of potency (dl-isoproterenol greater than l-epinephrine greater than l-norepinephrine) typical of a beta 2-adrenergic receptor. Binding was shown to be stereospecific with l-stereoisomers being more potent than their corresponding d-stereoisomers in causing half-maximal inhibition. Isoproterenol stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in a concentration-dependent manner, maximal levels (1.130 +/- 0.358 pmol cAMP/10(8) cells) being four times the basal levels. The results demonstrate the existence of a large number of beta-adrenergic receptors on baboon erythrocyte ghosts.
Cation channels from Tetrahymena cilia incorporated into planar lipid bilayers
Authors: Oosawa Y. , Sokabe M.
Published: December 2017

A single cation channel from Tetrahymena cilia was incorporated into planar lipid bilayers. This channel selected for K+, Na+, and Li+ over Cl- and gluconate-, and its single channel conductance (at +25 mV) was 211 +/- 8 pS (mean +/- SE) in 100 mM K+-gluconate. The channel was not voltage dependent and may contribute to the resting K+ conductance of ciliary membrane.
Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel
Authors: Eggena P. , Ma C. L. , Fahrenholz F. , Schwartz I. L.
Published: December 2017

The effects of a photoaffinity label for arginine vasopressin receptors, [Phe2, Phe(p-N3)3]AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of our previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.
Effects of mucosal sodium removal on cell volume in Necturus gallbladder epithelium
Authors: Davis C. W. , Finn A. L.
Published: December 2017

Necturus gallbladder epithelium transports sodium and chloride by a process that first involves the cellular entry of each ion across the apical membrane in an electrically silent process. In this paper we present results from cell volume and fluid flux measurements in the presence of different inhibitors and at normal and reduced sodium concentrations, which bear on the process by which ionic entry is effected. We find that reduction of mucosal sodium to a concentration of 10 mM has no effect on either cell volume or on the rate of transepithelial fluid transport, whereas the complete removal of sodium causes a significant decrease in cell volume in addition to its known inhibitory effect on fluid transport. Amiloride had no effect on cell volume at normal sodium concentrations but markedly reduced it when the sodium concentration was reduced to 10 mM. Amiloride, bumetanide, and dipyridamole markedly and reversibly inhibited fluid transport. Finally, the addition of ouabain to the serosal medium induced cell swelling, which was prevented by the removal of potassium from the mucosal medium. These results indicate that the process of sodium entry at the apical membrane is complicated and likely includes both cotransport (NaCl or Na-K-2Cl) and parallel exchange (Na-H and Cl-HCO3) transport mechanisms, and that the proportion of NaCl transported by the different mechanisms varies with the conditions.
ATP concentration gradients in cytosol of liver cells during hypoxia
Authors: Aw T. Y. , Jones D. P.
Published: December 2017

The activities of two ATP-requiring systems with different subcellular localizations were studied in cells in which average cellular ATP concentration was varied. The cytosolic ATP-sulfurylase activity varied linearly with the cellular ATP concentration; however, the plasma membrane Na+-K+-ATPase was substantially more sensitive to decreased ATP concentration. Under conditions where the cellular ATP concentration was lowered to 40% of control, Rb+ uptake was nearly zero. The results indicate that ATP-utilizing enzymes located in the plasma membrane in liver cells are exposed to a lower ATP concentration than are enzymes present in the cytosolic fluid surrounding the mitochondria. Analysis of radial diffusion of ATP from mitochondria, assuming that mitochondria are spherical and ATP consumption is zero order in ATP concentration, shows that the average ATP supply radius decreases as mitochondrial ATP production decreases. Hence, during limited ATP supply, enzymes with a greater average distance from mitochondria (e.g., plasma membrane ATPases) experience a more dramatic decrease in ATP concentration than enzymes in close proximity to mitochondria (e.g., cytoplasmic enzymes). Thus microheterogeneity of ATP supply can occur in cells without membranal compartmentation due to nonuniform distribution of ATP-generating and ATP-consuming systems.
Effects of pertussis toxin treatment on metabolism in hamster brown adipocytes
Authors: Schimmel R. J. , McCarthy L. , Dzierzanowski D.
Published: December 2017

This communication reports the effects of the exotoxin of Bordetella pertussis (pertussis toxin) on hamster brown fat cells. Pertussis toxin significantly increased the lipolytic and respiratory responses to isoproterenol but did not increase the basal rates of either of these processes. In contrast, the stimulation of respiration by the alpha-adrenergic agent phenylephrine was not altered by pertussis toxin. The inhibitory effects of adenosine on stimulated lipolysis, respiration, and adenylate cyclase activity were completely abolished by pertussis toxin, as was the ability of methylxanthines or adenosine deaminase to potentiate isoproterenol stimulation of respiration or lipolysis. These effects of pertussis toxin were associated with an ADP ribosylation of a single membrane protein having a molecular weight of approximately 41. These data demonstrate that pertussis toxin can prevent the inhibitory action of adenosine on brown fat cells and suggest that the effects of the nucleoside on these cells results from inhibition of adenylate cyclase. We further suggest that the enhanced responses to isoproterenol in pertussis-treated adipocytes results from a blockade of the action of endogenous adenosine. In addition to blocking adenosine action, pertussis toxin also abolished the antilipolytic effect of insulin. However, because the antilipolytic effect of insulin was prevented by adenosine deaminase and 3-isobutyl-1-methylxanthine and restored by 2-chloroadenosine, we conclude that insulin action on these cells is dependent on adenosine. Thus pertussis toxin blockade of insulin action appears to be secondary to blockade of adenosine action.
Kinetics of Cl-dependent K influx in human erythrocytes with and without external Na: effect of NEM
Authors: Kaji D. , Kahn T.
Published: December 2017

The majority of the ouabain-insensitive K influx in human erythrocytes is dependent on the presence of Cl. Recent studies have shown that a portion of the Cl-dependent K influx persists in the absence of external Na (Nao). It has been suggested that this Nao-independent component represents (K + Cl) cotransport, whereas the remainder of the Cl-dependent K influx seen on addition of external Na represents (Na + K + 2Cl) cotransport. In the present studies, the kinetics of Cl-dependent K influx were examined in the presence and absence of external Na, by varying external K and external Cl. Our studies suggest that the Nao-independent Cl-dependent pathway has a relatively low affinity for external K (Km 17-30 mM) in contrast to the high affinity of the Nao-augmented component (Km 3-4 mM). N-ethylmaleimide (NEM) stimulates the maximal velocity of the Nao-independent Cl-dependent K influx achievable without alteration of intracellular solutes but does not alter its Km for external K. In contrast, NEM has no stimulatory effect on the Nao-augmented component. The Cl dependence of the Nao-independent K influx is best described by a relatively flat curve with a mild upward concavity. The kinetic properties of the Nao-independent component of Cl-dependent K transport are very similar to those of the putative (K + Cl) cotransport pathway seen in low-K sheep erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
System for automatic activation of skinned muscle fibers
Authors: Chiu Y. C. , Quinlan J. , Ford L. E.
Published: December 2017

A system is described for automatically changing the solutions surrounding skinned muscle fibers at a controlled temperature. The system consists of a complex muscle chamber and solenoid valve-controlled vacuum-driven syringe pumps. The chamber has a muscle trough, a conduit for circulating coolant, and channels for precooling solutions. After traveling through the cooling channels, the solutions are injected into one end of the muscle trough and removed by suction at the other. The volume of the trough (approximately 40 microliter) is kept as low as possible to minimize the amount of solution required for complete solution exchange (approximately 400 microliter). Solution changes are complete within 400 ms. The timing of the sequence of solution changes is precisely controlled by digital timers that activate the solenoid valves of the pumps. By exposing the fibers briefly to a high concentration of free calcium and then changing to a buffered calcium solution, it is possible to achieve steady tension levels in 1-2 s, even when partially activating the fibers. Such rapid partial activation is possible because the exposure to free calcium can be precisely timed. The timers also allow the solution changes to be synchronized with other perturbations.
Atrial natriuretic factor inhibits Na-K-Cl cotransport in teleost intestine
Authors: O'Grady S. M. , Field M. , Nash N. T. , Rao M. C.
Published: December 2017

Addition of atrial natriuretic factor (ANF) to the contraluminal side of the intestinal mucosa of a marine teleost, the winter flounder Pseudopleuronectes americanus, inhibits short-circuit current, net transepithelial fluxes of Na and Cl, and the unidirectional influx of Rb across the brush border membrane. This action of ANF is closely mimicked by addition of 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP). In contrast to the intestine, the opercular epithelium of the flounder did not respond to the in vitro addition of either ANF or 8-BrcGMP. Because intestinal salt and water absorption diminishes when marine fish enter water of lower salinity, ANF may be an important hormonal regulator through which euryhaline fish adapt to varying salinities.
Interactions of pH and K+ conductance in cultured bovine retinal pigment epithelial cells
Authors: Keller S. K. , Jentsch T. J. , Koch M. , Wiederholt M.
Published: December 2017

Passive ion transport properties were studied in confluent monolayers of cultured bovine retinal pigment epithelial cells using intracellular microelectrode technique. The mean stable intracellular (designated by subscript i) potential was -59.1 +/- 0.8 (SE) mV. Extracellular (designated by subscript o) acidification induced a depolarization, whereas alkalinization induced a hyperpolarization. These effects were observed both in bicarbonate-free as well as in HCO3- Ringer (pHo changed by varying [HCO3-]o at constant pCO2). Acidification of pHi (changed by addition and removal of butyrate, CO2 or NH3) also caused a depolarization. Complete removal of HCO3-/CO2 at constant pHo caused a hyperpolarization. K+ transference, checked by applying high K+o, increased with K+o. It decreased with both extra and intracellular acidification and increased with alkalinization. In the presence of Ba2+, voltage reactions to changes in either pHo or pHi were greatly reduced. Depolarization by 40 mM K+ caused a similar reduction. It is suggested that K+ conductance of bovine retinal pigment epithelial cells is reduced by either intra- or extracellular acidification at normal [K+]o. Depolarization by high K+ induces an increase in K+ transference and reduces pH sensitivity.
Submaximal sodium-lack contractures in rapidly perfused frog ventricular strips
Authors: Shepherd N.
Published: December 2017

Rapid perfusion of strips of frog ventricular muscle (width less than or equal to 8 mm) by sodium-free Ringer yielded contractures with very fast rates of rise (half time approximately equal to 2 s) and flat plateaus. Tension in lithium Ringer was 0.64 (0.38-0.77) of the maximal tension the muscle could produce [i.e., for an extracellular potassium concentration ([K]o) of 160 mM, and an [Na]o of 0 mM at less than 15 degrees C], showing that intracellular calcium concentration ([Ca]i) in zero [Na]o and normal [K]o was insufficient to saturate the myofilaments. Tension was related to [Ca]o in an S-shaped manner (apparent Km = 1.3 microM), was rapidly increased by electrical depolarization, high potassium, or low temperature, but was unaffected by up to 10(-5) M dihydroouabain. From these data was derived the working hypothesis that the value of [Ca]i during the contracture plateau is a steady-state value due to influx through a sodium-dependent mechanism and calcium uptake or efflux via a sodium-independent mechanism. Also, 1 microM dihydroouabain increased peak twitch tension by 60% but did not potentiate tension during the plateaus of contractures obtained by partial replacement of Nao. This result supports the working hypothesis and suggests that the potentiating effect of raising Nai may depend on the membrane potential (D. A. Eisner, W. J. Ledener, and R. D. Vaughan-Jones. J. Physiol. Lond. 335: 723-743, 1983).
New amiloride analogue as hapten to raise anti-amiloride antibodies
Authors: Kleyman T. R. , Rajagopalan R. , Cragoe E. J. , Erlanger B. F. , Al-Awqati Q.
Published: December 2017

A new amiloride analogue, "amiloride-caproic acid," was synthesized, coupled to albumin, and used as a hapten to raise anti-amiloride antibodies in rabbits. The antibodies were affinity purified with an amiloride affinity column and characterized. Binding studies using [3H]benzamil showed a dissociation constant of 0.8 nM. Amiloride and amiloride-caproate inhibited [3H]benzamil binding; epsilon-guanidinocaproic acid showed no inhibition. Anti-amiloride antibodies reversed the inhibition by amiloride of sodium transport across toad urinary bladder. Anti-amiloride antibodies and an amiloride affinity column should provide useful tools for the characterization of the epithelial sodium channel.
Inositol-1,4,5-trisphosphate injection mimics fertilization potentials in sea urchin eggs
Authors: Slack B. E. , Bell J. E. , Benos D. J.
Published: December 2017

The effects of inositol-1,4,5-trisphosphate (IP3) and of diacylglycerol (DAG) and its analogues on the membrane potential of eggs from the sea urchin Strongylocentrotus purpuratus were examined. Injection of IP3 into eggs resulted in a change in membrane potential that was similar in magnitude and time course to the fertilization potential elicited by sperm attachment. In low-calcium seawater, IP3 injection elicited a partial response. DAG and its analogues phorbol myristyl acetate and 1-oleoyl-2-acetylglycerol did not affect membrane potential either when applied by perfusion or when injected. The results indicate that IP3, but not DAG or its analogues, may be involved in the generation of the fertilization potential triggered by the interaction of sperm with sea urchin eggs.
Role of calcium and calmodulin in release of kallikrein and tonin from rat submandibular gland
Authors: Maitra S. R. , Carretero O. A. , Smith S. W. , Rabito S. F.
Published: December 2017

We investigated the role of calcium and calmodulin as intracellular mediators of kallikrein and tonin release induced by norepinephrine (NE). We studied the secretion rate of kallikrein and tonin from submandibular gland of rat in response to NE in the presence or absence of calcium, two calcium blockers, and four different calmodulin antagonists. Submandibular gland slices were incubated in vitro, and glandular kallikrein and tonin secreted into the incubation medium were determined by direct radioimmunoassays and expressed as nanograms per minute per milligram tissue. NE (10(-5) and 10(-4) M) increased the kallikrein secretion from the control value of 8.2 +/- 2.6 to 134.9 +/- 41.4 (P less than 0.05) and to 191.2 +/- 62.7 (P less than 0.05), and the release of tonin from a basal rate of 3.5 +/- 0.6 to 51.5 +/- 9.1 (P less than 0.05) and to 64.4 +/- 13.7 (P less than 0.05). The deletion of calcium and addition of EGTA into the incubation medium significantly attenuated the secretion of kallikrein and tonin induced by NE. Nifedipine, at concentrations which inhibit voltage-dependent calcium channels, did not affect the release of kallikrein and tonin, and only a high concentration (10(-4) M) reduced the release. TMB-8, a blocker of intracellular calcium, had no effect either. Phenothiazines, triflupromazine (10(-6) M) and trifluoperazine (10(-4) M), decreased significantly the kallikrein release elicited by 10(-5) M NE.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolic differences between norepinephrine- and K+-stimulated anococcygeus muscle
Authors: Davidheiser S.
Published: December 2017

The effects of norepinephrine (NE) and K+ stimulation on aerobic glycolysis (JLac), O2 utilization (JO2), and isometric force of isolated rat anococcygeus smooth muscle were investigated to determine the relationship between metabolism and contractility. A large sustained increase in JLac was measured during NE stimulation, but only a small rise in JLac occurred during high-KCl depolarization. JO2, in both cases, correlated with the force response. The difference in JLac could not be explained solely on the lower force output of K+-depolarized muscles or on increased glycogenolysis during NE stimulation, but was perhaps due to activation of energy-requiring processes associated with receptor- rather than depolarization-mediated Ca2+ mobilization. When muscles were stimulated with a K2SO4 (substituted for NaCl) Krebs-Ringer bicarbonate (KRB) rather than a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium, JLac was significantly elevated due to an increased intracellular pH as a result of the low external Cl-. Lowering the pH of KRB or adding the anion exchange inhibitor 4-4'-diisothiocyanostilbene-2,2'disulfonic acid prevented the rise in JLac and also restored the initial fast component of the force response to NE in Cl(-)-free solution.
Effect of intraventricular pressure on protein synthesis in arrested rat hearts
Authors: Xenophontos X. P. , Gordon E. E. , Morgan H. E.
Published: December 2017

A modification of the Langendorff technique for perfusion of rat hearts was developed to allow a constant intraventricular pressure to be imposed on the left ventricle of arrested-catheterized hearts. This model was used to assess effects of increased aortic (60 and 120 mmHg) and intraventricular (0 and 25 mmHg) pressures on the rate of protein synthesis between 70 and 130 min of perfusion and on contents of ATP and creatine phosphate (creatine-P) in ventricles. Rates of protein synthesis in atria also were measured. Increased intraventricular or aortic pressure elevated the rate of protein synthesis (41%) compared with hearts supplied an aortic pressure of 60 mmHg and an intraventricular pressure of 0 mmHg. Higher intraventricular pressure also decreased the ribosomal subunit content. No change in the rate of protein synthesis was observed when intraventricular pressure was raised in hearts supplied an aortic pressure of 120 mmHg. Rates of atrial protein synthesis and contents of ATP and creatine-P were unchanged by elevations of either intraventricular or aortic pressure. These experiments indicate that stretch of the ventricular wall due to higher intraventricular or aortic pressure accelerated protein synthesis by maintaining the in vivo balance between rates of peptide chain initiation and elongation.
Catabolism of intracellular protein: molecular aspects
Authors: Beynon R. J. , Bond J. S.
Published: December 2017

All living cells regulate the content and composition of their resident proteins, but the mechanisms by which this is accomplished are not understood. The process of protein degradation has an important role in determining steady state and fluctuations of protein concentrations in mammalian cells. This process may be regulated by innate properties of the protein substrates, by factors that interact or "brand" proteins for degradation or by the degradative machinery of the cell. For a specific protein, there appears to be a committed step, an irreversible event that leads to rapid and extensive degradation. That initial event may or may not involve 1) proteolysis, 2) a nonproteolytic covalent modification or branding event (e.g., oxidation, ubiquitin conjugation), 3) denaturation or unfolding of the protein, or 4) sequestration. The degradative machinery of cells may either recognize proteins committed to degradation or initiate degradation, but the process must be selective because there is great heterogeneity in the rates of degradation for different proteins of one cell. The degradative process certainly requires proteases, and it is probable that lysosomal and extralysosomal proteases are involved in the catabolism of cellular proteins. We review here briefly what is currently known about the factors that may determine the half-life of a protein in a mammalian cell, the role of the protein substrate and sequestration in the process, the proteolytic and nonproteolytic enzymes that may initiate the degradative process, and the regulation of extensive degradation of proteins in cells.
Effects of Ca2+ and furosemide on Cl- transport and O2 uptake in rat parotid acini
Authors: Nauntofte B. , Poulsen J. H.
Published: December 2017

Stimulation-induced changes in Cl- content and O2 consumption of collagenase-isolated rat parotid acini were measured. In less than 10 s, carbachol caused a net Cl- efflux, corresponding to approximately 50% of the Cl- content, and increased the O2 uptake by 100%. The increase was inhibitable by ouabain and was dependent on the presence of extracellular Ca2+. Furosemide reduced the unstimulated 36Cl- uptake and prevented the reuptake of Cl- after carbachol-induced release. This suggests that a cotransport system is operating in both the unstimulated and stimulated states. Furthermore, furosemide inhibited the stimulated ouabain-sensitive O2 uptake. Raising intracellular Ca2+ by the calcium ionophore A23187 evoked the same pattern of Cl- loss and O2 uptake as carbachol. Our results are compatible with the following hypothesis. Carbachol raises intracellular Ca2+, causing an increased Cl- permeability of the luminal membrane, resulting in a net Cl- efflux. A subsequently enhanced influx of Cl- and Na+ via a furosemide-sensitive cotransport system increases intracellular Na+. This stimulates the Na+-K+-ATPase and thereby the O2 consumption.
Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts
Authors: Espinoza B. , Wharton W.
Published: December 2017

Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition in the growth of sparse human fibroblasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3',5'-cyclic monophosphate concentrations.