bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Na+ and K+ contents in rat thymocytes
Authors: Senn N. , Garay R. P.
Published: December 2017

A modified nystatin technique allowed the investigation of the initial rate of Na+ efflux as a function of internal Na+ content under steady-state conditions in rat thymocytes. This kinetic study showed that 1) ouabain-sensitive Na+ efflux as a function of internal Na+ can be adjusted by a three-sites kinetic model, with a maximal pump rate of 581 +/- 79 mmol.l cells-1.h-1 and an apparent dissociation constant for internal Na+ of 10.0 +/- 1.0 mmol/l cells (mean +/- SE of 3 experiments), 2) bumetanide-sensitive Na+ efflux was extremely low compared with the pump efflux (approximately 1%), and 3) ouabain- and bumetanide-resistant Na+ efflux was almost a linear function of internal Na+ content (as expected for a Na+ leak). This “all-pump” mechanism of thymocyte Na+ regulation was confirmed by non-steady-state experiments showing that 1) ouabain induced a rapid net Na+ gain and K+ depletion in fresh thymocytes and completely blocked the recovery of normal cation contents in Na+-loaded-K+-depleted thymocytes, and 2) bumetanide was unable to modify thymocyte Na+ and K+ contents. Na+ extrusion by Na+-loaded thymocytes was unaffected by prostaglandin E2, isoproterenol, or platelet-aggregating factor (PAF) and was slightly impaired in the adult spontaneously hypertensive rat of the Okamoto strain (10% lower rate constant for net Na+ extrusion, P less than 0.05). Concerning cell Na+ regulation, our results do not support the concept that rat thymocytes are more representative of vascular cells than enucleated erythrocytes.
Kinetics of Na-Li exchange in high and low K sheep red blood cells
Authors: Ryu K. H. , Adragna N. C. , Lauf P. K.
Published: December 2017

The kinetic parameters and transport mechanism of Na-Li exchange were studied in both low K (LK) and high K (HK) sheep red blood cells with cellular Na [( Na]i) and Li concentrations [( Li]i) adjusted by the nystatin technique (Nature New Biol. 244: 47-49, 1973 and J. Physiol. Lond. 283: 177-196, 1978). Maximum velocities (Vm) for Li fluxes and half-activation constants (K1/2) for Li and Na of the Na-Li exchanger were determined. The K1/2 values for both Li and Na appeared to be similar in both cell types, although they were about two to three times lower on the inside than on the outside of the membrane. Furthermore, the K1/2 values for Li were at least an order of magnitude smaller than those for Na, suggesting substantial affinity differences for these two cations. The Vm values for Li fluxes, on the other hand, appear to be lower in HK than in LK cells. When Na and Li fluxes were measured simultaneously, a trans stimulatory effect by Na on Li fluxes was observed. From measurements of Li influx at different concentrations of external Li and different [Na]i, the ratio of the apparent Vm to the apparent external Li affinity was calculated to be independent of [Na]i for both types of sheep red blood cells. Similar trans effects of external Na were observed on Li efflux at varying [Li]i. These results are expected for a system operating by a “ping-pong” mechanism.
Ca2+ induces charybdotoxin-sensitive membrane potential changes in rat lymphocytes
Authors: Grinstein S. , Smith J. D.
Published: December 2017

There is disagreement regarding the existence of Ca2+-activated K+ channels in lymphocytes. Depolarization, hyperpolarization, or little change in membrane potential (Em) has been reported following elevation of free cytosolic Ca2+ concentration ([Ca2+]i). Patch-clamping studies have demonstrated inhibition of voltage-gated K+ channels, but Ca2+-activated K+ channels have not been detected. We used charybdotoxin (CTX), a potent inhibitor of Ca2+-activated K+ channels, to assess their presence in rat thymic lymphocytes. Fluorescent probes were used to measure Em and [Ca2+]i in cell suspensions treated with ionomycin. At basal [Ca2+]i, CTX had no effect on Em, suggesting that Ca2+-activated K+ channels do not contribute importantly to the resting potential. Elevation of [Ca2+]i in the submicromolar range induced a hyperpolarization that was dependent on the outward K+ gradient. The shape and duration of the Em change closely followed the elevation of [Ca2+]i. This hyperpolarization was inhibited by nanomolar concentrations of CTX. When [Ca2+]i approached or exceeded 1 microM, a biphasic Em change was recorded. A transient, CTX-sensitive hyperpolarization was followed by a sustained depolarization. The latter was greatly reduced when external Na+ was omitted. The data suggest that thymic lymphocytes possess Ca2+-sensitive K+ channels, which are activated by moderate increases in [Ca2+]i, resulting in hyperpolarization. At higher [Ca2+]i, the effect of K+ channels on Em is superseded by opening of nonselective cation channels, producing depolarization. Variations in the level of [Ca2+]i attained in earlier studies can explain existing discrepancies.
Mechanism of hypoxic injury to pulmonary artery endothelial cell plasma membranes
Authors: Block E. R. , Patel J. M. , Edwards D.
Published: December 2017

We exposed monolayer cultures of pulmonary artery endothelial cells or plasma membranes derived from these cells to hypoxic (0 and 5% O2) and normoxic (20% O2; control) conditions and measured cellular contents of malondialdehyde and conjugated dienes, plasma membrane fluidity and lipid composition, and plasma membrane-dependent transport of 5-hydroxytryptamine (5-HT). Hypoxia caused significant increases in malondialdehyde and conjugated dienes, in fluidity, and in 5-HT transport. Hypoxia also caused a significant decrease in plasma membrane total phospholipids and a marked increase in plasma membrane free fatty acids that appeared to be due to release of fatty acids from the plasma membrane phospholipids. The increases in fluidity and 5-HT transport and the alterations in fatty acids were reversible after return to control conditions. These results indicate that hypoxia alters the physical state, lipid composition, and function of endothelial cell plasma membranes by a combination of stimulation of membrane lipid peroxidation and accelerated degradation of membrane phospholipids, the latter probably secondary to activation of membrane phospholipases.
Conductive pathways for HCO3- in basolateral membrane of salamander intestinal cells
Authors: White J. F.
Published: December 2017

To understand the route of HCO3- exit across the basolateral membrane of Amphiuma small intestinal cells during active H+ secretion the influence of bath HCO3- on serosal membrane potential (Vs) was measured using conventional microelectrodes. The villus sheet preparation was used, which allowed direct access to the basal membrane for microelectrode impalement. When tissues were incubated in Cl(-)-free (SO2-4 based) medium, Vs averaged -85.8 mV. Elevation of serosal bath [HCO3-] at constant CO2 increased Vs; reduction of the [HCO3-] reduced Vs and increased the fractional resistance of the serosal membrane. When serosal bath HCO3- and CO2 were completely replaced, Vs was -44.8 mV. The serosal membrane was also depolarized on complete replacement of medium HCO3- and CO2 when bath pH was buffered with phosphate. 4,4'-Dinitro-2,2'-stilbene disulfonate (DNDS) and acetazolamide blocked the decline in Vs produced by lowering serosal medium [HCO3-]. Replacement of serosal Na+ reduced Vs 32.7 mV. DNDS blocked this response. It is concluded that a Na+-dependent, stilbene-sensitive HCO3- exit pathway resides in the basolateral membrane of the intestinal cells. Possibly there is also a parallel, conductive pathway for HCO3-.
Flufenamic acid senses conformation and asymmetry of human erythrocyte band 3 anion transport protein
Authors: Knauf P. A. , Spinelli L. J. , Mann N. A.
Published: December 2017

With Cl as substrate, the human red blood cell anion transport (band 3) protein can exist in four conformations: Ei, with the transport site facing the cytoplasm; Eo, with the transport site facing the external medium; and ECli and EClo, the corresponding forms loaded with Cl. Flufenamic acid (FA), an inhibitor that binds to an external site different from the transport site, binds to Eo with a dissociation constant of 0.0826 +/- 0.0049 (SE) microM. Binding of iodide or sulfate to the external-facing transport site reduces the affinity by 1.66 or 14.3-fold, respectively. Changing from Eo to Ei lowers the affinity by 3.7-fold, and binding of cytoplasmic iodide to Ei further decreases the affinity by 5.5-fold. Thus changes in orientation of the transport site and substrate binding, even at the opposite side of the membrane, cause sufficient conformational changes in band 3 to affect FA binding substantially. If the possible effects of Cl binding to the transport site on FA affinity are estimated from the iodide data, the dependence of FA inhibitory potency on Cl concentrations inside and outside the cell suggests that there are at least 6.5 times as many inward-facing as outward-facing Cl-loaded transport sites. This information can be used to calculate the distribution of capnophorin among the various conformations under different circumstances and to devise conditions for recruiting the transport molecules toward a particular conformation.
Differential blockade of agonist- and depolarization-induced 45Ca2+ influx in smooth muscle cells
Authors: Wallnofer A. , Cauvin C. , Lategan T. W. , Ruegg U. T.
Published: December 2017

ATP stimulated 45Ca2+ influx in rat aortic smooth muscle cells in a concentration-dependent manner (EC50 = 3.6 +/- 0.5 X 10(-7) M). ADP and GTP were less effective than ATP in stimulating 45Ca2+ influx; AMP was weakly active and the adenosine agonist 5'-(N-ethyl-carboxamido)-adenosine (NECA) had no effect. ATP gamma S was about equieffective with ATP, whereas alpha,beta-methylene-ATP (APCPP) did not induce 45Ca2+ influx. Stimulation of 45Ca2+ influx by ATP was not abolished by the dihydropyridine Ca2+ channel antagonist darodipine (PY 108-068), which completely blocked depolarization-induced 45Ca2+ influx. Inorganic cations (La3+, Cd2+, Co2+, Ni2+, Mn2+, and Mg2+) were able to inhibit both agonist- and depolarization-induced 45Ca2+ influx. Cd2+, however, was approximately 20 times more selective in blocking K+-stimulated than agonist-stimulated 45Ca2+ influx. These data indicate that ATP-stimulated Ca2+ influx in rat aortic smooth muscle cells is resistant to darodipine but is reduced by La3+, Cd2+, and other inorganic blockers of Ca2+ channels.
ZO-1 and cingulin: tight junction proteins with distinct identities and localizations
Authors: Stevenson B. R. , Heintzelman M. B. , Anderson J. M. , Citi S. , Mooseker M. S.
Published: December 2017

The relative localization of ZO-1 and cingulin, the only two known components of the tight junction, was compared in Madin-Darby canine kidney (MDCK) cells, chicken small intestine, rat kidney distal convoluted tubule, and a hepatoma cell line. Immunoblot analysis demonstrated that cingulin and ZO-1 are immunologically unrelated and that, in the colon, cingulin is a single polypeptide with a molecular mass of 140 kDa. Immunofluorescent localization of cingulin and ZO-1 in confluent monolayers of MDCK cells showed identical staining patterns. However, subconfluent MDCK cells showed distinct localizations of the two proteins. Both cingulin and ZO-1 were found at the plasma membrane only at areas of cell-cell contact, but cingulin was diffusely distributed within the cytoplasm, whereas ZO-1 showed a more clustered internal arrangement. Cingulin and ZO-1 were identically localized at the plasma membrane of hepatoma tissue culture (HTC) cells at sites of cell-cell contact. In chicken intestine examined at the ultrastructural level, immunogold particles associated with cingulin were found approximately three times farther from the junctional membrane than those affiliated with ZO-1.
Demonstration of gap junctions in frog skin epithelium
Authors: Shahin S. H. , Blankemeyer J. T.
Published: December 2017

The morphology and distribution of the intercellular junctions were investigated in isolated skin of Rana pipiens using various electron-microscopic techniques. Our evidence demonstrates the presence of gap junctions and suggests that the distribution of gap junctions is not homogeneous among the epithelial strata. Gap junctions were less frequent in the stratum corneum and stratum granulosum than in the stratum spinosum and stratum germinativum. These results support a model of widespread intercellular coupling, although the lower number of gap junctions in the stratum granulosum suggests a possible deficiency in intercellular coupling. Tight junctions were found only in two apical strata of the epithelium (stratum corneum and stratum granulosum). Desmosomes were located in all strata.
Halothane-sensitivity gene and muscle contractile properties in malignant hyperthermia
Authors: Gallant E. M. , Mickelson J. R. , Roggow B. D. , Donaldson S. K. , Louis C. F. , Rempel W. E.
Published: December 2017

Malignant hyperthermia (MH) results from the presence of the halothane-sensitivity gene and is characterized by abnormalities in muscle function. Populations of genetically defined pigs were used to determine the in vivo and in vitro expression of this gene in both the homozygous and the heterozygous condition. On exposure to halothane, isolated muscle bundles from the homozygous halothane-sensitive pigs exhibited decreased tetanus tension and increased tetanus half-relaxation time and contracture and were clearly distinguished from homozygous normal muscles. The heterozygous and homozygous normal muscles were similar in contractile responses except for the occurrence of halothane-induced contractures in the heterozygotes. The heterozygous halothane-negative pigs did not exhibit the characteristic signs of an MH episode in response to halothane succinylcholine, although some metabolic responses were significantly altered (e.g., increased venous partial pressure of CO2 and arterial and venous K+ concentration). Thus the heterozygous pigs were not MH susceptible but did represent a phenotype distinct from the homozygous normal pigs both in vitro and in vivo. These data provide the first convincing evidence for the expression of the halothane-sensitivity gene in heterozygotes.
Administration of a creatine analogue induces isomyosin transitions in muscle
Authors: Moerland T. S. , Wolf N. G. , Kushmerick M. J.
Published: December 2017

A creatine analogue, beta-guanidinopropionic acid (beta-GPA), was administered in the food (2% wt/wt) and the water (0.5% wt/vol) of male CD-1 mice. Uptake of the phosphorylated analogue and depletion of phosphocreatine in hindlimb muscle was monitored by 31P nuclear magnetic resonance and was found to be complete within 7 wk. After this time, the isomyosin composition of soleus, extensor digitorum longus (EDL), and ventricle was analyzed by pyrophosphate gel electrophoresis. The analogue was found to induce significant alterations in the type of myosin expressed in soleus and EDL. Normal soleus contains both intermediate (IM) and slow (SM) myosins, and treatment reduced the relative content of IM by approximately 50%. In EDL, treatment decreased fast isomyosin FM3 by 60% compared with controls. Sodium dodecyl sulfate-gel electrophoresis also showed a decrease of parvalbumin in EDL by approximately 50%. Treatment had no significant effect on the isomyosin composition of heart ventricle. Levels of physical activity and concentrations of serum glucose and thyroxine of treated mice were not significantly different from controls. These results indicate a role for intracellular energetics in mediating adaptive changes in the phenotype of muscle in mature animals.
Daily running for 2 wk and mRNAs for cytochrome c and alpha-actin in rat skeletal muscle
Authors: Morrison P. R. , Biggs R. B. , Booth F. W.
Published: December 2017

The purpose of the study was to determine whether daily running durations that were 7-14% of the durations employed in the chronic stimulation protocols (consisting of 24 h of daily indirect electrical stimulation of skeletal muscles) still resulted in increases in a mitochondrial protein mRNA. Adult female rats were run 100 min/day on motor-driven treadmills for 2 wk. Documentation that rats underwent the stated training program was obtained by a 30-41% increase in citrate synthase activity in hindlimb muscles after 2 wk of the training. Cytochrome c mRNA was increased 17-56% in hindlimb muscles after the 2-wk training program. Thus shorter durations of exercise (100 min/day rather than 24 h/day) can increase cytochrome c mRNA. alpha-Actin mRNA increased 61-62% in fast-twitch muscles in the hindlimbs of the same rats that underwent the 2 wk of run training but did not increase in the predominantly slow-twitch soleus muscle. The increase in alpha-actin mRNA was unexpected, since it is well known that this type of physical exercise does not increase the size of fast-twitch skeletal muscle.
Role of calcium in mediating action of carbachol in T84 cells
Authors: Wong S. M. , Lindeman R. P. , Parangi S. , Chase H. S.
Published: December 2017

To examine the role of calcium in mediating carbachol's action in secretory epithelia, we simultaneously measured intracellular free [Ca] [( Ca]i) and transepithelial chloride transport in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value [( Ca]'i) normalized to control. Chloride transport was measured as the short-circuit current (Isc) with a voltage clamp. Monolayers were pretreated with cyclic AMP to augment the response of Isc to carbachol, a procedure that did not qualitatively change the response of the monolayer to carbachol. The carbachol-induced changes in Isc appeared to be dependent on the increase in [Ca]i. First, carbachol caused both Isc and [Ca]'i to increase in parallel. Isc increased from 32 +/- 5 to 70 +/- 9 microA and then declined to 57 +/- 16 microA while [Ca]'i increased from 72 +/- 14 to 156 +/- 22 nM and then declined to 133 +/- 45 nM. Second, the carbachol-induced increases in Isc and [Ca]'i were correlated. The greater the hormone-stimulated rise in [Ca]'i, the higher the increase in Isc. Third, carbachol's stimulation of Isc was blunted by preventing the calcium spike with the cellular calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Although the carbachol-induced increase in [Ca]'i appeared necessary for the increase in Isc, it was not clear if carbachol's action was solely the result of an increase in [Ca]'i. Increasing [Ca]'i with ionomycin, although causing Isc and [Ca]'i to increase in parallel, failed to increase Isc to the levels observed with carbachol. These experiments suggest that although the carbachol-induced increase in Isc is dependent on the increase in [Ca]i, the hormone may activate a second process that increases the sensitivity of the calcium-activated transport process to changes in [Ca]i.
cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture
Authors: Sáez J. A. , Gregory W. A. , Watanabe T. , Dermietzel R. , Hertzberg E. L. , Reid L. , Bennett M. V. L. , Spray D. C.
Published: December 2017

Pages C1–C11: J. A. Sáez, W. A. Gregory, T. Watanabe, R. Dermietzel, E. L. Hertzberg, L. Reid, M. V. L. Bennett, and D. C. Spray. “cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.” Page C8: Fig. 8B should appear as follows:
Chemical modification as an approach to elucidation of sodium pump structure-function relations
Authors: Pedemonte C. H. , Kaplan J. H.
Published: December 2017

Chemical modification of specific residues in enzymes, with the characterization of the type of inhibition and properties of the modified activity, is an established approach in structure-function studies of proteins. This strategy has become more productive in recent years with the advances made in obtaining primary sequence information from gene-cloning technologies. This article discusses the application of chemical modification procedures to the study of the Na(+)-K(+)-ATPase protein. A wide array of information has become available about the kinetics, enzyme structure, and various conformational states as a result of the combined use of inhibitors, ligands, modifiers, and proteolytic enzymes. We will review a variety of reagents and approaches that have been employed to arrive at structure-function correlates and discuss critically the limits and ambiguities in the type of information obtained from these methodologies. Chemical modification of the Na(+)-pump protein has already provided a body of data and will, we anticipate, guide the efforts of mutagenesis studies in the future when suitable expression systems become available.
[35S]ATP gamma S binding sites in the purified heart sarcolemma membrane
Authors: Zhao D. Y. , Dhalla N. S.
Published: December 2017

Purified heart sarcolemma membranes were found to bind a slowly hydrolyzable analogue of ATP [35S-labeled adenosine 5'-(gamma-thio)triphosphate [( 35S]ATP gamma S)] in a specific manner and exhibited two apparent affinity sites. The high-affinity site had a dissociation constant (KD) of 4.7-8.3 nM [maximum binding (Bmax) = 9.5-18.4 pmol/mg protein], whereas the low-affinity site had a KD of 655-1,257 nM (Bmax = 812-2,955 pmol/mg protein). Like ATP, other nucleotides such as GTP, UTP, ITP, and CTP were effective in displacing [35S]ATP gamma S binding. Although crude membrane preparations from different tissues also exhibited both high- and low-affinity sites for [35S]ATP gamma S, KD values for the high affinity sites were severalfold higher than that for the purified heart membranes. It is proposed that the high-affinity binding site for nucleotides may represent the ATP receptor in the heart cell membrane.
Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia
Authors: Atisook K. , Carlson S. , Madara J. L.
Published: December 2017

Glucose alters absorptive cell tight junction structure and, as deduced from an impedance analysis model, diminishes tight junction resistance in the small intestine (J.R. Pappenheimer, J. Membr. Biol. 100: 137-148, 1987; and J.L. Madara and J.R. Pappenheimer, J. Membr. Biol. 100: 149-164, 1987). Here we provide further evidence in support of this hypothesis using the conventional approach of analysis of mucosal sheets mounted in Ussing chambers. This approach offers advantages for investigating underlying mechanisms, including the effects of ions and inhibitors on the regulation of intercellular junctions by glucose. We show that phlorizin blocks a resistance decrease elicited by glucose and demonstrate that substitution of choline for sodium also prevents the response. The dilatations in absorptive cell tight junctions that accompany this glucose-elicited response are similarly prevented by phlorizin exposure or sodium substitution. The effects of phlorizin on junctional permeability can also be demonstrated in vivo. Phlorizin reduces the transjunctional flux of creatinine in glucose-perfused intestines of anesthetized animals, even when account is taken of the reduction of fluid absorption caused by phlorizin. Last, in vivo perfusion studies suggest that although, at 25 mM luminal glucose, virtually all glucose absorption is transcellular, at a luminal glucose concentration of 125 mM approximately 30% of glucose absorption occurs paracellularly because of solvent drag across tight junctions of altered permeability.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence for chloride secretion in the intestine of the winter flounder
Authors: O'Grady S. M. , Wolters P. J.
Published: December 2017

The effects of a novel antisecretory peptide (CAP) isolated from porcine heart and vasoactive intestinal peptide (VIP) on ion transport were investigated in the winter flounder intestine. Partially purified CAP caused a two- to sixfold increase in the serosa-negative short-circuit current (Isc) with no significant change in tissue conductance. CAP significantly inhibited the serosal-to-mucosal (S-M) unidirectional Cl flux without affecting either Na or Rb transepithelial fluxes. The Isc after the addition of CAP was completely inhibited by 0.1 microM atriopeptin III (AP-3), 10 microM bumetanide, and 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). In contrast to the effects of CAP on Isc, VIP decreased the serosa-negative Isc by 40-60%. VIP stimulated the S-M unidirectional Cl flux without affecting transepithelial Na transport. When food was present in the intestine, the basal Isc was occasionally found to be serosa positive, ranging between 10 and 40 microA/cm2. Treatment of tissues exhibiting serosa-positive currents with VIP resulted in an increase (positive direction) in Isc. Addition of CAP to tissues with a serosa-positive Isc or to tissues pretreated with VIP resulted in a serosa-negative Isc.(ABSTRACT TRUNCATED AT 250 WORDS)
Stimulation of glucose transport in Clone 9 cells by exposure to alkaline pH
Authors: Ismail-Beigi F. , Mercado C. L. , Loeb J. N.
Published: December 2017

Incubation of a rat liver cell line (Clone 9) for 2 h at pH 8.5 was found to result in a profound (5- to 8-fold) stimulation of cytochalasin B-inhibitable glucose transport. The enhancement of glucose transport after exposure to elevated external pH (achieved by lowering the CO2 tension in a bicarbonate-containing medium) was demonstrable within 15 min, was half-maximal at pH 8.0, and was near-maximal at pH 8.6. Intracellular pH rose linearly with incremental changes in external pH, from pH 7.45 to 8.6 with a slope of 0.6. The increase in transport activity in response to incubation at alkaline pH was accompanied by a parallel increase in lactate production and persisted for more than an hour after external pH was restored to normal. During the latter period, intracellular glucose concentration (less than 10% of that of the external medium under control conditions) increased greater than 10-fold to approximate that in the extracellular medium. Incubation of these cells at pH 8.5 for 2 h resulted in a complete resistance of cell ATP levels to challenge with 5 mM cyanide, suggesting that the adaptive facilitation of glucose transport was of sufficient magnitude to permit a marked stimulation of glycolytic ATP synthesis on inhibition of oxidative phosphorylation. The enhancement of glucose transport was attributable to an increase in the maximum velocity (Vmax) rather than to any change in the Michaelis constant (Km) for transport and was not prevented by cycloheximide. It is concluded that the marked stimulation of glucose transport resulting from exposure of these "low-glucose" cells to alkaline pH reflects either an increase in the abundance of functional glucose transporters in the plasma membrane or an increase in their catalytic turnover rate.
cGMP-dependent protein kinase mediates the reduction of Ca2+ by cAMP in vascular smooth muscle cells
Authors: Lincoln T. M. , Cornwell T. L. , Taylor A. E.
Published: December 2017

The major action of forskolin, the diterpine activator of adenylate cyclase, in primary (unpassaged) rat aortic smooth muscle cells is to reduce vasopressin-stimulated Ca2+ concentrations. In repetitively passaged cells, however, forskolin by itself increased Ca2+ levels by apparently stimulating Ca2+ uptake into the cell and had much smaller effects on inhibiting vasopressin-stimulated Ca2+ elevations. Both primary and passaged smooth muscle cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase was greatly reduced or absent in passaged smooth muscle cells. The introduction of purified cGMP-dependent protein kinase into the cytoplasm of passaged cells prevented forskolin from elevating intracellular Ca2+ and restored the capacity of forskolin to reduce vasopressin-stimulated Ca2+ mobilization. Similar effects were observed for isoproterenol in passaged smooth muscle cells. When introduced into cells, the active catalytic subunit of the cAMP-dependent protein kinase did not lead to reductions in Ca2+ levels. These results suggest that cAMP elevations lead to profound changes in Ca2+ metabolism through activation of both cAMP- and cGMP-dependent protein kinases. Activation of cGMP-dependent protein kinase by cAMP leads to the reduction in intracellular Ca2+, whereas activation of cAMP-dependent protein kinase may only mediate the uptake of Ca2+ from extracellular sources.
Halothane-dependent release of intracellular Ca2+ in blood cells in malignant hyperthermia
Authors: Klip A. , Mills G. B. , Britt B. A. , Elliott M. E.
Published: December 2017

The concentration of ionized cytosolic calcium [( Ca2+]i) was determined in peripheral blood mononuclear cells from normal and malignant hyperthermia (MH)-susceptible humans and pigs, using the fluorescent Ca2+ indicator indo-1. [Ca2+]i was slightly but significantly elevated in cells from MH human cells relative to normal cells (198 +/- 18 nM, n = 15, and 146 +/- 14 nM, n = 11, respectively, P less than 0.05). Anesthetic concentrations of halothane in the cell suspension resulted in a rapid increase in [Ca2+]i in cells from both normal and MH humans or pigs. The increases (delta) were more pronounced in cells from MH subjects than from normal individuals (delta at 5.7 mM halothane: 245 +/- 53 vs. 57 +/- 11 nM, respectively) and from MH than from normal pigs (delta of 241 +/- 63 vs. 53 +/- 27 nM, respectively). Removal of extracellular Ca2+ obliterated the delta[Ca2+]i caused by halothane in cells from normal humans or pigs but only decreased by about half the delta[Ca2+]i in cells from MH humans or pigs. In 1,2-bis-(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-loaded cells, in the absence of extracellular Ca2+, halothane failed to increase [Ca2+]i. This suggests that buffering Cai2+ with BAPTA precludes detection of release of Ca2+ from intracellular stores, explaining the previous observations made with quin2, a highly chelating Ca2+ indicator. It is concluded that clinical concentrations of halothane allow influx of Ca2+ in cells from both normal and MH-susceptible individuals but release Ca2+ from intracellular stores selectively in cells from the latter group.(ABSTRACT TRUNCATED AT 250 WORDS)
Thyroid hormone induction of Na(+)-K(+)-ATPase and its mRNAs in a rat liver cell line
Authors: Gick G. G. , Ismail-Beigi F.
Published: December 2017

The expression of mRNAs encoding the alpha- and beta-subunits of Na(+)-K(+)-ATPase (Na(+)-K+ pump) was examined in a rat liver cell line, Clone 9, in various thyroidal states. Northern blot analysis of total RNA isolated from cells incubated in hypothyroid serum-containing medium revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1-(mRNA alpha 1) and beta- (mRNA beta) subunits; mRNAs encoding the alpha 2- and alpha 3-subunits were undetectable. There was a discrepancy in the abundance of mRNA alpha 1 relative to mRNA beta such that mRNA alpha 1 exceeded the sum of the multiple mRNA beta bands by approximately 35-fold. 3,3',5-Triiodothyronine (T3) produced a coordinate augmentation of mRNA alpha 1 and mRNA beta contents that was demonstrable within 2 h and preceded the stimulation of Na(+)-K(+)-ATPase activity. After incubation of cells with T3 for 48 h, Na(+)-K(+)-ATPase activity was stimulated by 1.32-fold, whereas mRNA alpha 1 and mRNA beta abundances were increased 1.46- and 2.87-fold, respectively. Treatment of cells for 6 h with 10 micrograms/ml cycloheximide, a concentration sufficient to inhibit protein synthesis by 95%, elicited a 3.5- and 5.1-fold increase in mRNA alpha 1 and mRNA beta content, respectively. Cycloheximide abrogated the stimulatory effect of T3 on mRNA beta abundance, whereas the T3-induced increase in mRNA alpha 1 content was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of elevated temperature on protein breakdown in muscles from septic rats
Authors: Hall-Angeras M. , Angeras U. , Hasselgren P. O. , Fischer J. E.
Published: December 2017

Elevated temperature has been proposed to contribute to accelerated muscle protein degradation during fever and sepsis. The present study examined the effect of increased temperature in vitro on protein turnover in skeletal muscles from septic and control rats. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham operated. After 16 h, the extensor digitorum longus (EDL) and soleus (SOL) muscles were incubated at 37 or 40 degrees C. Protein synthesis was determined by measuring incorporation of [14C]phenylalanine into protein. Total and myofibrillar protein breakdown was assessed from release of tyrosine and 3-methylhistidine (3-MH), respectively. Total protein breakdown was increased at 40 degrees C by 15% in EDL and by 29% in SOL from control rats, whereas 3-MH release was not affected. In muscles from septic rats, total and myofibrillar protein breakdown was increased by 22 and 30%, respectively, at 40 degrees C in EDL but was not altered in SOL. Protein synthesis was unaffected by high temperature both in septic and nonseptic muscles. The present results suggest that high temperature is not the primary mechanism of increased muscle protein breakdown in sepsis because the typical response to sepsis, i.e., a predominant increase in myofibrillar protein breakdown, was not induced by elevated temperature in normal muscle. It is possible, however, that increased temperature may potentiate protein breakdown that is already stimulated by sepsis because elevated temperature increased both total and myofibrillar protein breakdown in EDL from septic rats.
Replacement of molecular species of phosphatidylcholine: influence on erythrocyte Na transport
Authors: Engelmann B. , Op den Kamp J. A. , Roelofsen B.
Published: December 2017

The phosphatidylcholine-specific transfer protein (PC-Tp) from bovine liver was used to replace endogeneous erythrocyte phosphatidylcholine (PC) with various amounts of five different molecular species of PC. Furosemide-sensitive (FS) Rb uptake, Na-Li exchange, and Na-K pump rates were considered in relation to the nature and extent of those replacements. Changes in fatty acid contents of PC after incorporation of different molecular species fell within a variation range (10-30%) similar to that found in large populations of healthy individuals. Di16:0-PC accelerated Na-Li exchange and FS Rb uptake by approximately 40 and 25%, respectively. Some reduction (20%) in FS Rb uptake was seen in 16:0/18:2-PC-enriched erythrocytes. Incorporation of 16:0/22:6-PC accelerated Na-Li exchange and FS Rb uptake by greater than 40 and 20%, respectively. Apart from inhibitory effects of 16:0/18:1-PC and di16:0-PC (24 and 19%, respectively) the Na-K pump rate was virtually unchanged by incorporation of different PC molecular species. Exogeneous PC molecules are exclusively inserted in the outer membrane leaflet and, particularly in the case of di16:0-PC, migrate slowly to the cytoplasmic leaflet. Prolonged incubation of cells (up to 21 h) after replacement with di16:0-PC showed that both Na-Li exchange and FS Rb uptake rates responded differently to redistribution of newly inserted molecules over both bilayer halves. Compared with cells exhibiting a selective incorporation of di16:0-PC in the outer monolayer, additional enrichment with disaturated species in the inner monolayer accelerated FS Rb uptake, whereas Na-Li exchange rate reverted to control values. It is concluded that small changes in fatty acid composition of PC induced by limited replacement of phospholipid molecular species can cause considerable changes in Na-Li exchange rate and FS Rb uptake. Differences in phospholipid molecular species composition could contribute to known interindividual variability of both Na-Li exchange and Na-K cotransport.
Alterations of bile acid and bumetanide uptake during culturing of rat hepatocytes
Authors: Follmann W. , Petzinger E. , Kinne R. K.
Published: December 2017

Uptake by the multispecific bile acid transport system of [3H]taurocholate, [14C]cholate, and [3H]-bumetanide into primary cultures of rat hepatocytes was compared with their uptake into freshly isolated rat hepatocytes. The uptake maximum velocity (Vmax) of all compounds declined in primary culture, whereas the Michaelis constant (Km) values remained stable. Loss of uptake was not due to the reduction of driving forces as evaluated from the level of ATP and the activity of Na(+)-K(+)-ATPase. No alpha-fetoprotein was detectable in culture supernatants. Neither growth factors (glycylhistidyl-lysine, epidermal growth factor), peroxisome and cell proliferators (nafenopin, dimethyl sulfoxide), nor bile acids prevented the loss of transport in hepatocyte culture. However, addition of dibutyryl adenosine 3'5'-cyclic monophosphate protracted the transport activity significantly. When cultured rat hepatocytes with reduced transport were detached by trypsin, cells rounded up and showed the same uptake capacity for bumetanide, cholate, and taurocholate as seen in freshly isolated hepatocytes. "Cryptic" transport activity in the lower basolateral membrane facing the support was found using an incubation chamber for cultured hepatocytes, which allowed us to distinguish simultaneously between uptake via the upper and lower basolateral membrane of the cultured cells.
Protection by glycine of proximal tubules from injury due to inhibitors of mitochondrial ATP production
Authors: Weinberg J. M. , Davis J. A. , Abarzua M. , Kiani T. , Kunkel R.
Published: December 2017

We have determined whether glycine or glutathione can protect rabbit proximal tubules damaged by chemical inhibitors of oxidative phosphorylation: antimycin A, rotenone, cyanide, oligomycin, or carbonyl cyanide m-chlorophenylhdrazone (CCCP). All the agents severely depleted cell ATP levels within 15 min and caused lethal cell injury, as quantified by lactate dehydrogenase (LDH) release. Glycine and glutathione largely prevented this injury without altering the primary effects of the inhibitors on tubule respiration or the depletion of ATP. Buthionine sulfoximine and 1,3-bis(2-chloroethyl)-1-nitrosourea decreased cell glutathione but did not prevent the protective effects of either glycine or glutathione in tubules treated with rotenone. Protection was sustained during both a 15-min exposure and a 45-min postwash period irrespective of whether the wash removed the agent or mitochondrial function recovered. Cysteine uniquely induced a dramatic recovery of mitochondrial function in tubules washed after treatment with CCCP. These data 1) demonstrate that the cytoprotective effects of glycine previously seen during hypoxia extend to other tubule lesions characterized by severe ATP depletion, 2) emphasize the actions of glycine to preserve cell structural integrity in spite of sustained severe impairment of ATP-generating processes in proximal tubules, and 3) indicate that it is glycine rather than intracellular or extracellular glutathione which mediates protection.
Basolateral K conductance: role in regulation of NaCl absorption and secretion
Authors: Dawson D. C. , Richards N. W.
Published: December 2017

In this review we explore the possible role of basolateral K conductance (gK) in the regulation of salt absorption and secretion. This inquiry is prompted by a growing body of evidence which, taken together, suggests that basolateral gK is very labile and that alterations in basolateral gK may be a key feature in both stimulatory and inhibitory regulatory mechanisms. We first consider the role of basolateral gK in relation to models for salt absorption and secretion, particularly in relation to the maintenance of cellular charge balance and the obligatory coupling between the apical and basolateral membranes that is produced by transcellular current flow. Next, we review some of the experimental evidence that suggests that changes in basolateral gK are associated with transport regulation. The cellular mechanisms that are known to impact on K channel regulation are considered in a general way, and finally, we consider the use of integrated models for understanding possible coordinate regulation of apical and basolateral cell membranes.
Sarcomere dynamics during isotonic velocity transients in single frog muscle fibers
Authors: Granzier H. L. , Mattiazzi A. , Pollack G. H.
Published: December 2017

If the load on a tetanized fiber is abruptly changed to a new steady value, the ensuing fiber length change shows the well-known "isotonic velocity transient," in which the velocity oscillates before settling at some steady value. We studied sarcomere dynamics during these transients using two methods: optical diffraction and a segment-length method. Our principal aim was to determine whether these transients might be a reflection of the fact that sarcomere shortening is often found to be stepwise. We found that pauses in sarcomere shortening occurred during the low-velocity phases of the transient and that steps of sarcomere shortening occurred during the high-velocity phases. Thus the isotonic transient appears to arise from the steps. In addition to the isotonic transient, we studied the well-known isometric transient, in which fiber length is abruptly changed, and ensuing tension response is measured. Again, we found that the transient may be a reflection of the stepwise shortening pattern.
Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells
Authors: Thorens B. , Cheng Z. Q. , Brown D. , Lodish H. F.
Published: December 2017

The "liver" isoform of the facilitated diffusion glucose transporter is expressed predominantly in liver, intestine, kidney, and pancreatic islet beta-cells. The apparent molecular mass of the transporter in liver, kidney, and intestine is different, as detected by Western blot analysis of membrane proteins using antipeptide antibodies. However, as assessed by Northern blot analysis and molecular cloning, the same mRNA is expressed in these tissues, indicating that there are tissue-specific posttranslational modifications of the same transporter polypeptide. As determined by immunofluorescence analysis on frozen tissue sections, the liver glucose transporter is present on the sinusoidal membrane of hepatocytes, on the basolateral membrane of fully differentiated absorptive intestine epithelial cells, and on the basolateral membrane of proximal tubule cells of the kidney nephron. This localization is consistent with the involvement of the liver glucose transporter in several key steps of glucose metabolism: glucose uptake and release by the liver and absorption or reabsorption by epithelial cells of the intestine and kidney, respectively.
Muscarinic regulation of potassium transport in a human submandibular epithelial cell line
Authors: Ship J. A. , Patton L. L. , Wellner R. B.
Published: December 2017

Results of previous studies suggest that the transport of K+ by salivary ducts is under muscarinic control. The mechanisms by which this regulation occurs have not been well defined, however. In this paper, we describe mechanisms involved in the muscarinic regulation of K+ (86Rb) transport in HSG-PA, an epithelial cell line derived from human submandibular gland duct. Stimulation of HSG-PA cells by carbachol, a muscarinic agonist, increases both 86Rb influx and efflux, which results in a decrease in the equilibrium content of 86Rb within the cells. Increases in both fluxes are dose dependent with respect to carbachol concentration, and both responses can be blocked by atropine, a muscarinic antagonist. The carbachol-stimulated 86Rb fluxes appear to be calcium dependent since 1) the calcium ionophore A23187 increases 86Rb fluxes in these cells, 2) cells loaded with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA; a calcium chelator) exhibit a reduced ability to respond to carbachol stimulation, and 3) removal of extracellular calcium concentration reduces the carbachol-stimulated effects. Treatment of HSG-PA cells with 10(-7) M phorbol myristate acetate (PMA) partially blocks the carbachol-stimulated changes in 86Rb fluxes, suggesting that protein kinase C plays a role in this response. PMA also partially blocks A23187-stimulated 86Rb influx, suggesting that activation of protein kinase C inhibits muscarinic-stimulated K+ influx by blocking either the Ca2+ signal (X. He, X. Wu, and B.J. Baum. Biochem. Biophys. Res. Commun. 152: 1062-1069, 1988), steps subsequent to this effect, or both.(ABSTRACT TRUNCATED AT 250 WORDS)
A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells
Authors: Venglarik C. J. , Bridges R. J. , Frizzell R. A.
Published: December 2017

We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-[cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl)oxy]acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.
Effect of temperature on myosin phosphorylation in mouse skeletal muscle
Authors: Moore R. L. , Palmer B. M. , Williams S. L. , Tanabe H. , Grange R. W. , Houston M. E.
Published: December 2017

The effect of muscle contraction on phosphorylatable myosin light chain (P-light chain) phosphate content and isometric twitch tension was examined at 25, 30, and 35 degrees C in intact mouse extensor digitorum longus muscle. Peak tetanic tension was unaffected by temperature, whereas peak unpotentiated isometric twitch tension was inversely proportional to muscle incubation temperature. The extent of phosphate incorporation into P-light chain elicited by a 20-s train of twitches (5/s) was inversely proportional to muscle incubation temperature, whereas the fractional increase in twitch tension (twitch potentiation) elicited by repetitive stimulation was directly proportional to muscle incubation temperature. After the twitch train, the rate of decline of potentiated twitch tension and of P-light chain dephosphorylation was directly proportional to muscle incubation temperature. The net result was that a significant and unique relationship between P-light chain phosphate content and contraction-induced tension potentiation existed at each temperature examined. The slope of the P-light chain phosphate vs. isometric twitch potentiation relationship varied directly as a function of muscle incubation temperature. The observations that the slope of this relationship increases and that unpotentiated twitch tension decreases when muscle incubation temperature is increased support the hypothesis that contraction-induced tension potentiation in intact mammalian skeletal muscle is the result of a sensitization of the contractile element to activation by Ca2+ that is brought about by P-light chain phosphorylation.