bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrical properties and morphology of single vascular smooth muscle cells in culture
Authors: Toro L. , Gonzalez-Robles A. , Stefani E.
Published: December 2017

Single vascular smooth muscle cells (VSMC) were isolated from the caudal artery and vein and studied after 2 or 3 days in culture. Current clamp with intracellular microelectrodes and "whole-cell" voltage-clamp techniques were used. Also, scanning and transmission electron microscopy studies were performed, revealing morphological characteristics of smooth muscle in culture. Cells could contract in response to electrical and chemical stimuli. The passive membrane properties recorded with intracellular microelectrodes in a mammalian saline were as follows: 1) for artery, resting potential Vm = -56 +/- 5 mV (mean +/- SD), input resistance Rin = 590 +/- 35 M omega, membrane time constant tau m = 19 +/- 2 ms, membrane capacity C/cm2 = 1.3 +/- 0.2 microF/cm2, and length constant lambda = 900 +/- 40 micron; and 2) for vein, Vm = -66 +/- 3 mV, Rin = 450 +/- 25 M omega, tau m = 19 +/- 2 ms, C/cm2 = 1.0 +/- 0.1 microF/cm2, and lambda = 1,300 +/- 200 micron. The values calculated for a short cable and the observed change of the membrane potential as a single exponential, in response to hyperpolarizing pulses of current, both indicate that the cell membrane behaves as an isopotential surface. With hyperpolarizing pulses, both cell types gave linear voltage-current (V-I) relationships with a constant slope, Rin. On the other hand, depolarizing pulses elicited outward rectification. Voltage-clamp experiments show an outward voltage-dependent K+ current (IK) when the cell membrane is depolarized beyond approximately equal to -40 mV from holding levels approximately equal to -60 mV. Maximum slope conductances were of approximately 120 microS/cm2. Blocking of K+ channels with tetraethylammonium ions did not unmask an inward current. These results indicate that VSMC from rat caudal artery and vein in culture have K+ channels responsible for the graded depolarization of the cell membrane in response to an electrical stimulus. Furthermore, this experimental approach seems to be adequate to further study the electrical responses of VSMC from vessels at distinct stages of development, and to follow these responses as the cells change in a defined environment.
Importance of purine nucleotide cycle to energy production in skeletal muscle
Authors: Flanagan W. F. , Holmes E. W. , Sabina R. L. , Swain J. L.
Published: December 2017

The purpose of this study was to determine the role of the purine nucleotide cycle in aerobic energy production. Rats received either saline or 5-amino-4-imidazolecarboxamide riboside (AICAriboside), a precursor to an inhibitor of adenylosuccinate lyase (AICAR). Muscle tension was quantified during gastrocnemius stimulation, and muscle metabolite content was measured to obtain an estimate of the activity of the enzymes of the cycle. AICAriboside prevented the increase in synthetase and lyase activities observed in control animals during moderate (aerobic) stimulation, and was accompanied by marked muscle dysfunction. Although glycolytic energy production was not impaired in the AICAriboside-treated animals (lactate production occurred), total energy production did not meet energy demand. These results suggest that disruption of the purine nucleotide cycle impairs aerobic energy metabolism. Tetanic (anaerobic) stimulation produced more rapid fatigue in the AICAriboside-treated group. Total energy production was again impaired in the AICAriboside-treated animals, but lactate production was similar in both groups. These findings suggest the loss of the initial aerobic component of energy generation in tetanically stimulated muscle of AICAriboside-treated animals. The results of this study indicate that disruption of the purine nucleotide cycle at the level of the synthetase and lyase reactions is associated with skeletal muscle dysfunction, and suggest that the cycle plays an anapleurotic role in providing citric acid cycle intermediates that enhance aerobic energy production in contracting skeletal muscle.
Coupling of Na-H exchange and Na-K pump activity in cultured rat proximal tubule cells
Authors: Harris R. C. , Seifter J. L. , Lechene C.
Published: December 2017

Na-H exchange was studied using electron probe analysis and microfluorescent pH analysis of individual cells, in 3-day primary cultures of rat proximal tubule cells (RPTC) obtained from 40- to 50-day-old Sprague-Dawley rats. After Na-K pump inhibition, the initial rate of net Na influx was inhibited 87% by 1 mM amiloride. K influx, an estimate of Na-K pump activity, was increased approximately three times in cells containing high Na (0.114 mM K X mM P-1 X min-1) compared with control cells containing low Na (0.038 mM K X mM P-1 X min-1). Single cell measurements of RPTC loaded with the cytoplasmic pH indicator 5- (and -6) carboxy-4',5'-dimethylfluorescein indicated that there was reversible intracellular acidification in the absence of external Na or in the presence of amiloride. When intracellular acidification was induced by the addition and subsequent removal of NH4Cl, recovery of intracellular pH was inhibited in the absence of external Na or in the presence of amiloride. Using a similar protocol, it was found that after intracellular acidification, the rate of Na influx increased at least 5.9 times, and intracellular Na content was increased 3.15 +/- 0.64 times at 60 s. There was an initial 50% inhibition of Na-K pump activity within the first 60 s compared with control (nonacidified) RPTC, secondarily followed by an increase in Na-K pump activity. Amiloride (0.5 mM) inhibited the acidification-induced increase in Na influx, and persistent acidification led to a persistent inhibition of Na-K pump activity compared with control. In summary, these results demonstrate that Na-H exchange mediates the majority of net Na influx into RPTC under our basal conditions and is necessary for maintenance of intracellular pH homeostasis. In RPTC, Na-H exchange is further activated by intracellular acidification, leading to a net increase in intracellular Na content, which secondarily stimulates Na-K pump activity. The initial inhibition of Na-K pump activity may be due to a direct effect of intracellular acidification.
Activation of protein kinase C potentiates norepinephrine release from sinus node
Authors: Shuntoh H. , Tanaka C.
Published: December 2017

Localization of binding sites of [20-3H]phorbol-12,13-dibutyrate [( 3H]PDBu) and the involvement of Ca2+-phospholipid-dependent protein kinase (protein kinase C) in the release of norepinephrine (NE) from sympathetic nerve terminals in the guinea pig sinus node were investigated. There was a single class of specific [3H]PDBu binding sites in the heart. [3H]NE release from the sinus node preloaded with [3H]NE was evoked by electrical stimulation in superfusing medium containing Ca2+ or by the concomitant presence of Ca2+ ionophore and Ca2+, in Ca2+-free medium. 12-O-tetradecanoylphorbol-13-acetate (TPA) potentiated the evoked [3H]NE release. The effect of TPA was antagonized by both polymyxin B and H-7, inhibitors of protein kinase C. TPA increased the apparent affinities of electrical stimulation-evoked release for extracellular Ca2+. The possibility that protein kinase C plays a role in transmembrane signal transduction involved in the release of NE from peripheral adrenergic nerve terminals in the guinea pig sinus node warrants continued study.
3-O-methyl-D-glucose transport in tumoral insulin-producing cells
Authors: Malaisse W. J. , Giroix M. H. , Malaisse-Lagae F. , Sener A.
Published: December 2017

Tumoral insulin-producing cells of the RINm5F line were exposed at different temperatures, and for various lengths of time to increasing concentrations of 3-O-methyl-D-[U-14C]glucose. The uptake of the hexose represented a temperature-sensitive and saturable process, so that no rapid equilibration of hexose concentrations across the plasma membrane was reached, especially at low temperature and/or high concentrations of 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-[U-14C]glucose was not affected by a prior loading of the cells with the unlabeled hexose and its release from prelabeled cells was observed in the absence of any concentration gradient across the plasma membrane. The uptake of D-[U-14C]glucose and utilization of D-[5-3H]glucose was inhibited by 3-O-methyl-D-glucose, which failed, however, to affect D-[U-14C]glucose oxidation. At variance with the situation found in normal insulin-producing cells, the transport of D-glucose into the tumoral cells may thus play a regulatory role in its metabolism.
Ionomycin acts as an ionophore to release TRH-regulated Ca2+ stores from GH4C1 cells
Authors: Albert P. R. , Tashjian A. H.
Published: December 2017

In the GH4C1 strain of rat pituitary cells, ionomycin, a divalent cation ionophore, induces a rapid and transient spike in cytosolic free Ca2+ concentrations [( Ca2+]i) similar to that induced by the Ca2+-mobilizing hormone thyrotropin-releasing hormone (TRH). To test directly the hypothesis that ionomycin causes the spike in [Ca2+]i by altering cellular Ca2+ stores, we have measured ionomycin-induced changes in 45Ca2+ fluxes and have compared these to previously characterized changes induced by TRH. Ionomycin (half-maximal concentration = 30 nM) rapidly (within 1 min) induced a release into the medium of 50-60% of cell-associated 45Ca2+, paralleling the spike in [Ca2+]i. The ionomycin-induced 45Ca2+ efflux was greater than with TRH, and TRH did not induce further 45Ca2+ efflux in the presence of ionomycin. Ionomycin pretreatment blocked induction of the spike in [Ca2+]i elicited by TRH but did not alter basal or TRH-induced enhancement of inositol phosphate levels. These results provide evidence that the spike in [Ca2+]i induced by ionomycin or TRH is produced largely by release of Ca2+ into the cytosol from the same intracellular pool, followed by rapid extrusion of the released Ca2+ into the extracellular space. However, unlike TRH, ionomycin appears to release cellular Ca2+ directly, acting as an ionophore, without the generation of known second messengers.
Effects of Ca2+/Mg2+ removal on aiNa, aiK, and tension in cardiac Purkinje fibers
Authors: Chapman R. A. , Fozzard H. A. , Friedlander I. R. , January C. T.
Published: December 2017

Sheep cardiac Purkinje fibers were exposed to solutions free of divalent cations for hour-long periods, while intracellular Na+ and K+ activities were measured using ion-sensitive microelectrodes. Intracellular Na+ activity (aiNa) increased to 50.1 +/- 8.1 mM, and intracellular K+ activity (aiK) decreased to 76.7 +/- 3.5 mM. These ionic changes could be blocked by the presence of Mg2+ or the Ca2+ channel blocking agents D 600 and nifedipine. The rise in aiNa and the fall in aiK was accentuated by the inhibition of the Na+-K+ pump with acetylstrophanthidin or by removal of extracellular K+. These results demonstrate that in cardiac Purkinje fibers removal of divalent cations produces intracellular loading of Na+ by Na+ entry through the Ca2+ channel. On reexposure to Ca2+-containing solutions, the cells become loaded with Ca2+, and the fibers exhibit large contractures. These observations implicate Na+-Ca2+ exchange in the entry of Ca2+ into these cells during Ca2+ repletion and in the etiology of the calcium paradox.
Dynamic state of collagen: pathways of collagen degradation in vivo and their possible role in regulation of collagen mass
Authors: Laurent G. J.
Published: December 2017

There is now evidence that collagen turnover in normal tissues can occur at rapid rates. In skin of adult rats, the mean rate lies between 3 and 5%/day, but rates of greater than 10%/day have been reported in some tissues, such as lung and periodontal ligament. The major sites of this degradation are still uncertain. Intracellular degradation, which occurs within minutes of synthesis, may be extensive, but its magnitude varies between tissues. This process may have evolved to allow the adaptive advantages of rapid turnover without compromising the supportive role of fibrillar collagen. There is now abundant evidence that changes in collagen degradation play an important role in the regulation of collagen mass. Increased breakdown of extracellular collagens may be required whether the mass is increased or decreased. However, preliminary evidence suggests that the degradation of newly synthesized collagen may be up- or down-regulated, depending on the direction of the change in content.
Sodium-hydrogen exchange system in LLC-PK1 epithelium
Authors: Moran A.
Published: December 2017

Sodium influx into LLC-PK1 cells has been characterized. The main amiloride-sensitive pathway for sodium entry through the apical membrane in this cell line is sodium-hydrogen exchange with an apparent Km for sodium of 19 mM. Influx is pH dependent and is amiloride sensitive with K1/2 of 30 microM. Inhibition of the sodium transport by protons (at 1 mM external sodium) is consistent with an interaction of H+ ions at a single site having an apparent pKH of 7.2. These data are similar to those reported previously for brush border membrane (BBM) isolated from kidney proximal tubule. However, in contrast to previously published reports, H+ does not compete with amiloride in blocking Na+ influx and exhibits a mixed inhibition with sodium ions. The latter, together with a direct and independent assessment of amiloride and sodium interaction, indicates that amiloride does not compete with sodium for the same site. Possible explanations for the discrepancy in the literature concerning the interaction of Na+, H+, and amiloride with the Na+-H+ exchanger and the characteristics of the Na+-H+ exchange system in LLC-PK1 are discussed.
Cytosolic free calcium concentration and glucose transport in isolated cardiac myocytes
Authors: Cheung J. Y. , Constantine J. M. , Bonventre J. V.
Published: December 2017

The role of cytosolic free Ca2+ concentration, [Cai], in mediating insulin's stimulatory effect on glucose transport was investigated in isolated Ca2+-tolerant rat ventricular cells. Approximately 98% of glucose transport in isolated myocytes was inhibited by phloretin. Insulin-accelerated glucose transport by 50-115% over basal transport rate. Removal of extracellular Ca2+ had no effect on either the basal transport rate or insulin's stimulatory action, indicating that extracellular Ca2+ was not necessary for insulin's effect to be manifest. Addition of A23187 had no effect on glucose transport rate. Under basal conditions, [Cai] was 167 +/- 12 nM as measured by fura-2 fluorescence and 239 +/- 22 nM by null-point titration with arsenazo III. Loading cells with fura-2 did not affect basal glucose transport rates. In addition, the stimulatory effect of insulin on glucose transport was preserved in fura-2 loaded cells. In paired experiments, insulin did not increase [Cai] as measured by fura-2 fluorescence or null-point titration despite acceleration of glucose transport. In contrast, addition of KCl (40 mM) increased [Cai] from 168 +/- 30 to 287 +/- 51 nM and resulted in 50% reduction in glucose transport rate. In other experiments designed to control for the hyperosmolar effects of KCl, NaCl (40 mM) caused no change in [Cai] but also inhibited glucose transport rate by 50%. We conclude that an elevation in [Cai] is unlikely to be the intracellular signal mediating insulin's effect on glucose transport since insulin's stimulatory effect was not reduced by Ca2+ -free media, insulin had no detectable effect on [Cai], and elevation of [Cai] by KCl did not result in stimulation of glucose transport.
Energy metabolism of renal cell lines, A6 and MDCK: regulation by Na-K-ATPase
Authors: Lynch R. M. , Balaban R. S.
Published: December 2017

The energy metabolism of two continuous cell lines of renal origin, MDCK (Madin-Darby dog kidney) and A6 (toad kidney), was investigated by measuring the oxygen consumption (QO2) and lactate production (Jlac) by cells taken into suspension from monolayer cultures. Cells suspended from fully differentiated monolayers produce approximately 80% of their ATP requirements from oxidative metabolism. The interrelationship between ion transport and metabolism was determined by analyzing the ouabain sensitive components of intermediary metabolism under control conditions and after the stimulation of active Na-K transport with nystatin. In both cell lines, approximately 25% of the net rate of ATP production was inhibited by ouabain. Ouabain inhibited Jlac by 40% in MDCK and 45% in A6 cells, whereas QO2 was decreased by only 20% in both cell lines. In the presence of 0.05 mg nystatin/mg cell protein, ouabain sensitive Jlac increased by 75% in MDCK and was more than doubled in A6, whereas the ouabain-sensitive QO2 was not statistically different than control. This preferential stimulation of glycolysis with nystatin was not due to a limited capacity of mitochondrial oxidative phosphorylation since nystatin treatment of cells incubated without glucose (no glycolysis) significantly elevated the rate of QO2. These data demonstrate that aerobic glycolysis is more sensitive than is QO2 to changes in hydrolytic activity of the Na-K-adenosine triphosphatase (ATPase), in both cell lines.
Contraction of rat thoracic aorta strips induced by phorbol 12-myristate 13-acetate
Authors: Itoh H. , Lederis K.
Published: December 2017

Phorbol 12-myristate 13-acetate (PMA) induced a slow and progressive increase in tension of rat thoracic aorta strips in the presence of extracellular Ca2+. Complete relaxation could not be obtained in Ca2+-free buffer containing 1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) and 10(-7) M PMA. In the absence of extracellular Ca2+, PMA (10(-7) M) induced a small but sustained contraction which was not altered by the addition of another 2 mM EGTA and 3 X 10(-5) M verapamil. Papaverine (10(-4) M) relaxed the PMA-induced contraction to the base line, but phentolamine (10(-5) M), cyproheptadine (10(-5) M), atropine (10(-5) M) and tetrodotoxine (10(-6) M) did not change the contraction. Ca2+-depleted muscle strips, prepared by four repeated applications of 10(-7) M norepinephrine in Ca2+-free buffer, were contracted by 10(-7) M PMA, but at a lower maximum tension than nontreated strips. The action of PMA on rat aorta strips in Ca2+-free buffer did not require the presence of the adventitial layer or endothelial cells. These results suggest that PMA may induce activation of protein kinase C and smooth muscle contraction in the absence of extracellular Ca2+, without an increase in myoplasmic Ca2+.
Negative inotropic effect of extracellular calcium buffering in cardiac muscle
Authors: Shimoni Y. , Ginsburg S.
Published: December 2017

Heart muscle contracts more vigorously when calcium levels are raised. A transient depletion of calcium from restricted extracellular spaces occurs with each contraction. We decided to maintain the concentration of this ion at a constant level by using an external calcium buffering system. It was found that buffering calcium at a millimolar level (using citrate as a buffer) caused a decrease, rather than an increase in the strength of contraction. The mean reduction in peak tension was by 27% in guinea pig and by 50.5% in frog atrium. This finding is analyzed; its most plausible explanation is the hypothesis that the buffer dissipates a calcium inhomogeneity, consisting of a higher calcium concentration adjacent to the membrane. Alternative interpretations such as intracellular acidosis, were tested experimentally and ruled out.
Tissue distribution of rat S100 alpha and S100 beta and S100-binding proteins
Authors: Zimmer D. B. , Van Eldik L. J.
Published: December 2017

To understand the physiological role of the calcium-binding proteins S100 alpha and S100 beta, it is necessary to determine the distribution of these proteins and detect their intracellular targets in various tissues. The distribution of immunoreactive S100 alpha and S100 beta in various rat tissues was examined by radioimmunoassay. All tissues examined contained detectable S100, but the S100 beta/S100 alpha ratio in each tissue differed. Brain, adipose, and testes contained 18- to 40-fold more S100 beta than S100 alpha; skin and liver contained approximately equivalent amounts and kidney, spleen, and heart contained 8- to 75-fold more S100 alpha than S100 beta. Analysis of S100-binding proteins by gel overlay showed that each tissue possessed its own complement of binding proteins. The S100 beta-binding profile was indistinguishable from the S100 alpha-binding profile and both of these profiles were distinct from the calmodulin-binding profile. These observations suggest that the differential distribution and quantity of the individual S100 polypeptides and their binding proteins in various tissues may be important factors in determining S100 function.
Adenylate cyclase stimulation alters transport in frog retinal pigment epithelium
Authors: Hughes B. A. , Miller S. S. , Farber D. B.
Published: December 2017

The effect of adenylate cyclase inhibitors and activators on enzyme kinetics and cyclic AMP (cAMP) levels was determined in the bullfrog retinal pigment epithelium (RPE). The RPE enzyme has two Km for ATP: 5.2 X 10(-4) and 4.0 X 10(-5) M, with Vmax of 2.5 and 0.25 nmol X mg protein-1 X min-1. Forskolin, the most potent activator, produced a fourfold increase in enzyme activity and, in the presence of isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, caused a 20-fold increase in cAMP levels. Alloxan, a potent inhibitor, blocked the forskolin-induced activation of this enzyme. In the isolated RPE choroid, forskolin (plus IBMX) produced changes in membrane voltage and resistance that were similar in magnitude but slower in time course than those produced by exogenous cAMP. Like exogenous cAMP, forskolin also decreased steady-state fluid and solute transport in isotonic proportions. Therefore, modulation of RPE adenylate cyclase activity plays an important role in the control of RPE transport.
PO2-dexamethasone interactions in fibroblast growth and antioxidant enzyme activity
Authors: Randhawa P. S. , Hass M. A. , Frank L. , Massaro D.
Published: December 2017

Fetal rat lung fibroblasts were cultured in a gas phase of 20% O2, 5% CO2 (PO2 measured, 150 Torr) or 2% oxygen, 5% CO2 (PO2 measured, 25 Torr) with or without 100 nM dexamethasone (Dex). Superoxide dismutase (SOD) activity per cell increased spontaneously during 4 days of incubation at both PO2, but catalase (CAT) activity tended to fall during this time and glutathione peroxidase (GPx) activity showed no consistent trend during this interval. Cells cultured at a low PO2 had a lower protein content and SOD activity compared with air controls. Dex inhibited cell proliferation and enhanced intracellular accumulation of protein at the low PO2 but prevented the increase in protein content without affecting cell multiplication at a PO2 of 150 Torr. SOD activity per cell was enhanced by Dex at a low PO2 but reduced in 20% O2, 5% CO2. An increase in CAT and GPx activity per cell resulted on exposing fibroblasts to Dex in the presence of low PO2. These results show that Dex affects the growth and antioxidant enzyme activity of fetal lung fibroblasts, and this action of Dex can be modulated by changing the ambient PO2.
Cellular calcium regulates outward currents in rabbit intestinal smooth muscle cell
Authors: Ohya Y. , Kitamura K. , Kuriyama H.
Published: December 2017

The nature of transient and oscillatory outward currents (ITO and IOO) in fragmented smooth muscle cells (smooth muscle ball, SMB) from the longitudinal muscle layer of the rabbit ileum, was studied using a single electrode voltage clamp technique. With a high K+ solution containing 0.3 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) in the pipette and physiological salt solution (PSS) in the bath, the Ca inward current was followed by a large transient outward current (ITO) and spontaneous oscillations of the outward current (IOO) on the sustained outward current (ISO) were elicited by a depolarizing pulse, positive to -30 mV (holding potential of -60 mV). When the internal fluid of the SMB was replaced with Cs+-tetraethylammonium+ (TEA+) solution, or when the concentration of EGTA in the pipette was increased to 4 mM, using the intracellular perfusion technique, both ITO and IOO were abolished. In Mn2+ solution both currents were also inhibited. Bath application of TEA+, procaine or A23187 completely blocked both ITO and IOO. Caffeine (0.3-1 mM) enhanced the amplitude of ITO and generations of IOO, and concentrations of caffeine over 3 mM transiently enhanced, but finally suppressed both these currents. These results suggest that the generation of ITO is closely related to the Ca2+ influx, whereas the generation of IOO may be initiated by an increment in the intracellular concentration of Ca2+, possibly released from store sites.
Ca2+ and MgATP2- dependence of shortening in skinned single smooth muscle cells
Authors: Warshaw D. M. , McBride W. J. , Hubbard M. S.
Published: December 2017

Most studies of skinned smooth muscle have been performed in whole tissue preparations. In this study, we report the development of a chemically skinned single smooth muscle cell preparation from the toad, Bufo marinus, stomach. Isolated smooth muscle cells were skinned using saponin. The effect of various ionic environments (i.e., changing free Ca2+ and MgATP2-) on skinned cell contractile response was assessed by measuring cell lengths from populations of cells using a computer-assisted length-measuring system. Comparison of cell length histograms were used to determine the extent of cell shortening in response to a given ionic perturbation. Once skinned, the single cells shortened with a sensitivity to free calcium (ED50 = 1.5 microM Ca2+) that was three orders of magnitude lower than potassium depolarized cells (ED50 = 1.5 mM Ca2+). In addition to the calcium sensitivity, the effect of free MgATP2- on the extent of cell shortening was investigated. The extent of cell shortening was dependent on free MgATP2- with the maximum shortening response occurring at MgATP2- greater than 1 mM.
Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca
Authors: Levy L. A. , Murphy E. , London R. E.
Published: December 2017

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.
Lactate transport by cardiac sarcolemmal vesicles
Authors: Trosper T. L. , Philipson K. D.
Published: December 2017

L-lactate is taken up by cardiac sarcolemmal vesicles in a process that is saturable with respect to L-lactate, stereospecific, associated specifically with the sarcolemmal membrane, and inhibited by other monocarboxylic acids and by the protein modifiers p-chloromercuriphenyl-sulfonate and N-ethylmaleimide. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of the inorganic anion transporter, is without effect. The L-lactate transport is very sensitive to pH. Uptake is stimulated by a proton gradient directed inward and decreased when internal pH is lower than external pH. Passive diffusion of nonionized lactic acid into the vesicles is negligible at physiological pH and appears to remain minor even when external pH is lowered by more than one unit. Also, the mechanism does not require specific Na+-L-lactate contransport. The properties of the L-lactate transporting system in cardiac sarcolemmal vesicles appear similar to those of the monocarboxylate transporter in erythrocytes, hepatocytes, and Ehrlich ascites cells. The present results do not allow a distinction to be made between stepwise interaction of lactate- and H+ or association of nonionized lactic acid with the carrier.
Na microelectrode study of pathways of Na entry into Amphiuma intestinal absorptive cells
Authors: White J. F. , Ellingsen D. , Mayer S.
Published: December 2017

To define the pathways of Na+ entry into intestinal villus cells, intracellular Na+ activity (aiNa) was measured in Amphiuma duodenum using conventional and Na-sensitive microelectrodes. Replacement of Na+ in the luminal medium reduced aiNa rapidly; replacement of Na+ in the serosal medium caused a slow decline of aiNa. Hence, mucosal and serosal membranes are both permeable to Na+. Ouabain addition to the serosal medium caused aiNa to increase over 4 h. When Na+ was present only in the mucosal medium and Na+ transport was inhibited with ouabain, aiNa increased over 4 h. With galactose or valine (20 mM) in the mucosal medium aiNa was greater at 2 h relative to paired control tissues. The gain in aiNa was unaffected by replacement of luminal medium Cl- with gluconate or exposure to 1 mM furosemide or amiloride. Amiloride, at 1 mM, was detected by the Na-sensitive neutral carrier. Over a wide range of Na+ concentrations in the luminal medium the rate of Na+ entry across the mucosal membrane correlated strongly (r = 0.95) with the electrochemical gradient for Na+ across the luminal membrane. It is concluded that aiNa of urodele intestinal cells is maintained at a low level by the operation of a Na+-K+ pump. Na+ entry across the luminal membrane occurs by diffusion and by the cotransport with sugars and amino acids. Luminal NaCl cotransport and Na+-H+ exchange do not appear to contribute to Na entry to a measurable extent but it is possible that these transport processes operate at a slow rate, but were inhibited secondary to inhibition of the Na-K pump.
A gated 31P NMR study of tetanic contraction in rat muscle depleted of phosphocreatine
Authors: Shoubridge E. A. , Radda G. K.
Published: December 2017

Rats were fed a diet containing 1% beta-guanidino-propionic acid (GPA) for 6-12 wk to deplete their muscles of phosphocreatine (PCr). Gated 31P nuclear magnetic resonance (NMR) spectra were obtained from the gastrocnemius-plantaris muscle at various time points during either a 1- or 3-s isometric tetanic contraction using a surface coil. The energy cost of a 1-s tetanus in unfatigued control rat muscle was 48.4 mumol ATP X g dry wt-1 X s-1 and was largely supplied by PCr; anaerobic glycogenolysis was negligible. In GPA-fed rats PCr was undetectable after 400 ms. This had no effect on initial force generated per gram, which was not significantly different from controls. Developed tension in a 3-s tetanus in GPA-fed rats could be divided into a peak phase (duration 0.8-0.9 s) and a plateau phase (65% peak tension) in which PCr was undetectable and the [ATP] was less than 20% of that in control muscle. Energy from glycogenolysis was sufficient to maintain force generation at this submaximal level. Mean net glycogen utilization per 3-s tetanus was 78% greater than in control muscle. However, the observed decrease in intracellular pH was less than that expected from energy budget calculations, suggesting either increased buffering capacity or modulation of ATP hydrolysis in the muscles of GPA-fed rats. Our results demonstrate that the transport role of PCr is not essential in contracting muscle in GPA-fed rats. PCr is probably important in this regard in the larger fibers of control muscle. Although fast-twitch muscles depleted of PCr have nearly twice the glycogen reserves of control muscle, glycogenolysis is limited in its capacity to fill the role of PCr as an energy buffer under conditions of maximum ATP turnover.
Strontium, barium, and manganese metabolism in isolated presynaptic nerve terminals
Authors: Rasgado-Flores H. , Sanchez-Armass S. , Blaustein M. P. , Nachshen D. A.
Published: December 2017

To gain insight into the mechanisms by which the divalent cations Sr, Ba, and Mn affect neurotransmitter release from presynaptic nerve terminals, we examined the sequestration of these cations, in comparison to Ca, by mitochondrial and nonmitochondrial [presumably smooth endoplasmic reticulum (SER)] organelles and the extrusion of these cations from isolated nerve terminals. Sequestration was studied in synaptosomes made leaky to small ions by treatment with saponin; efflux was examined in intact synaptosomes that were preloaded with the divalent cations by incubation in depolarizing (K rich) media. The selectivity sequence for ATP-dependent mitochondrial uptake that we observed was Mn much greater than Ca greater than Sr much greater than Ba, whereas that for the SER was Ca greater than or equal to Mn greater than Sr much greater than Ba. When synaptosomes that were preloaded with divalent cations were incubated in Na- and Ca-free media, there was little efflux of Ca, Ba, Sr, or Mn. When the incubation was carried out in media containing Na without Ca, there was substantial stimulation of Ca and Sr efflux, but only slight stimulation of Ba or Mn efflux. In Na-free media, the addition of 1 mM Ca promoted the efflux of all four divalent cations, probably via Ca-divalent cation exchange. In summary, the sequestration and extrusion data suggest that, with equal loads, Mn will be buffered to the greatest extent, whereas Ba will be least well buffered. These results may help to explain why Mn has a very long-lasting effect on transmitter release, while the effect of Sr is much briefer.
Trypsin- and chymotrypsin-induced chemiluminescence by isolated rat glomeruli
Authors: Basci A. , Shah S. V.
Published: December 2017

We examined whether the generation of reactive oxygen metabolites (as quantified by measuring luminol-amplified chemiluminescence) by isolated rat glomeruli could be triggered enzymatically. No response was observed with thrombin (1 or 10 U/ml), collagenase (100, 200, or 400 U/ml), or plasmin (0.1 or 1 U/ml). In contrast, chymotrypsin and trypsin caused a dose-dependent (10-200 micrograms/ml) increase in chemiluminescence from glomeruli. The peak response with chymotrypsin (100 micrograms/ml) and trypsin (50 micrograms/ml) was as follows: resting, 16 +/- 2 X 10(3) cpm/mg protein, n = 17; chymotrypsin, 233 +/- 58 X 10(3) cpm/mg protein, n = 17; and trypsin, 221 +/- 38 X 10(3) cpm/mg protein, n = 10. Tubules had only a minor response. Soybean trypsin inhibitor and aprotinin caused marked inhibition, indicating the dependency of the chemiluminescence response on the protease enzyme activity. The chemiluminescence response was by glomeruli rather than by "contaminating" leukocytes, since a similar marked response (n = 6) was observed in glomeruli isolated from cyclophosphamide-treated leukopenic (leukocyte less than 1,000/mm3) rats. Superoxide dismutase, a scavenger of superoxide, and free-radical scavengers benzoate and tryptophan inhibited the glomerular chemiluminescence response to trypsin and chymotrypsin. Neutral proteases from infiltrating leukocytes and/or renal tissue have been shown to be released in glomerular diseases; our results, which show the generation of chemiluminescence in response to neutral proteases, suggest a potential mechanism for the production of reactive oxygen metabolites in glomerular diseases.
Covalent labeling of hydrosmotic toad bladder receptors with an antagonist of vasotocin
Authors: Eggena P. , Buku A. , Ma C. L. , Somoza L. I. , Wyssbrod H. R. , Schwartz I. L. , Glass J. D.
Published: December 2017

A photoreactive analogue of vasotocin, [1-desamino,4-lysine(azidobenzoyl),8-arginine]vasotocin (4-N3-AVT), has been examined in the isolated toad urinary bladder for biological activity and binding to hormonal receptors. Although 4-N3-AVT induced only a small increase in bladder permeability to water, it behaved as a potent inhibitor of hydrosmotic action of [8-arginine]vasotocin (AVT) and [8-arginine]vasopressin (AVP). The inhibitory action of 4-N3-AVT was readily reversed on removal of the analogue from the serosal bathing solution. On the other hand, when bladders were exposed to 4-N3-AVT in the presence of long wavelength UV light (365 nm), the inhibition by 4-N3-AVT was not reversed on washout of the analogue. The dose of vasopressin required for a half-maximal response (ED50 value) was increased from 5 X 10(-9) to 1.3 X 10(-7) M in bladders photolabeled with 4-N3-AVT and the maximal response capacity of the tissue (intrinsic activity) was reduced to 79% of nonphotolabeled controls. A crude membrane preparation derived from bladders photolabeled with 4-N3-AVT contained 72 fmol of specific binding sites for tritium-labeled vasopressin per milligram protein, whereas nonphotolabeled controls had 136 fmol of specific binding sites per milligram protein. These observations suggest that 4-N3-AVT forms a covalent bond with hydrosmotic receptors in the presence of UV light. This is the first antagonistic photoaffinity analogue observed in the toad bladder and it may serve as a useful tool for analyzing the cellular mechanism of action of antidiuretic hormone.
Multiple pathways for uptake of paraquat, methylglyoxal bis(guanylhydrazone), and polyamines
Authors: Byers T. L. , Kameji R. , Rannels D. E. , Pegg A. E.
Published: December 2017

The uptake of polyamines, methylglyoxal bis(guanylhydrazone) (MGBG), and paraquat [N,N-dimethyl-4,4'-bipyridylium] into control Chinese hamster ovary (CHO) cells and a mutant CHO cell line selected for resistance to the toxicity of MGBG was examined. In contrast to control CHO cells, the mutant cells had no detectable uptake of MGBG or any of the polyamines. There was no difference between the two cell lines in the uptake of alpha-aminoisobutyric acid (AIB), which indicates that there was no general change in membrane transport processes. The mutant cells were also found to be resistant to the toxicity of paraquat and to have a reduced capability to take up the herbicide. This finding confirms that the uptake of paraquat is necessary for the toxicity of this compound and that the paraquat is taken up by a transport system that also transports MGBG. Competition experiments showed that an excess of unlabeled paraquat inhibited uptake of MGBG and, to a lesser extent, uptake of putrescine and spermidine, but no inhibitory action on spermine uptake could be detected. Studies with type II cells isolated from rat lung also demonstrated uptake of paraquat and spermidine, but paraquat was only a weak inhibitor of spermidine uptake in this system. These results suggest that there may be multiple systems for the uptake of MGBG and polyamines and that paraquat is taken up by at least one but not by all of these systems.
Measurement of Na+-K+ coupling ratio of Na+-K+-ATPase in rabbit proximal tubules
Authors: Avison M. J. , Gullans S. R. , Ogino T. , Giebisch G. , Shulman R. G.
Published: December 2017

A combination of 23Na nuclear magnetic resonance (NMR) spectroscopy and a K+-selective electrode was used to make simultaneous measurements of net Na+ and K+ fluxes across plasma membranes of rabbit renal proximal tubules after an abrupt stimulation of Na+-K+-ATPase. After a step in extracellular K+ concentration ([K+]o) from low to higher concentration (0.1-0.3 mM to 0.5-5.2 mM) at 25 degrees C, net extrusion of Na+ and uptake of K+ were observed. These fluxes were completely inhibited by ouabain (10(-3) M). Because initial rates of K+ uptake in presence or absence of Ba2+ (a known inhibitor of plasma membrane K+ conductance) were indistinguishable, net K+ flux was virtually unidirectional. Because suspension buffers contained neither glucose nor amino acids and the ratio of net Na+ and K+ fluxes (JNa and JK, respectively) was constant over a wide range of transmembrane Na+ gradients and absolute values of the JNa and JK, it is likely that changes in electrogenic or passive net fluxes across plasma membranes were insignificant in the first 30–45 s after the [K+]o step. Thus the ratio of these initial net Na+ and K+ fluxes corresponds closely to the Na+-K+ coupling ratio of the Na+-K+-ATPase. In 12 experiments, the measured Na+-K+-ATPase coupling ratio was 1.54 +/- 0.07 (SE). The coupling ratio was constant over a wide range of intracellular Na+ content, intracellular sodium concentration, [K+]o and transmembrane Na+ gradient. The coupling ratio also remained constant over an eightfold range of Na+-K+-ATPase rates.
Intestinal absorptive cell tight junctions are linked to cytoskeleton
Authors: Madara J. L.
Published: December 2017

Permeation of intercellular tight junctions in epithelia may be altered by maneuvers that affect the cytoskeleton. Conversely, agents that alter tight-junction permeability also often produce alterations in cytoskeletal structure. However, anatomic links between the tight junction and the cytoskeleton have not been clearly defined. We explore the anatomy of the perijunctional cytoskeleton by applying electron microscopy to cytoskeletal preparations of whole intestinal absorptive cells using detergent extraction techniques. Individual elements of the perijunctional cytoskeleton, including actin microfilaments as determined by S1 labeling, appear to associate with the tight junction by means of plaque-like densities that intimately associate with the lateral membrane at the site of the tight junction. Furthermore, such associations are not diffuse within the tight junction, but occur only at sites of fusions ("kisses") between lateral membranes that are thought to represent the specific intrajunctional sites at which the barriers to transjunctional permeation reside. These data provide evidence of intimate cytoskeletal-tight-junction associations, which may represent the anatomical basis for cytoskeletal control of tight-junction permeability.
Mechanical work, oxygen consumption, and efficiency in isolated frog and rat muscle
Authors: Heglund N. C. , Cavagna G. A.
Published: December 2017

The total work done during shortening, in repeated stretch-shortening cycles and the subsequent recovery oxygen consumption were measured in isolated frog (Rana esculenta) sartorius at 12 degrees C and rat (Wistar strain) extensor digitorum longus (EDL) and soleus at 20 degrees C. Two procedures were followed. In the first, the muscles were lengthened in the relaxed state and stimulated isometrically just before and during the first part of shortening. The peak efficiency (positive work done divided by the energetic equivalent of the oxygen consumed) was approximately 25% at 0.75–1.5 muscle lengths/s (Lo/s) in sartorius, 19% at 1.0 Lo/s in EDL, and 15% at 0.5 Lo/s in the soleus. In contrast to the measured efficiency values, the ratio between the tension-time integral and the oxygen consumption (the economy) is greater in soleus than in EDL. In the second procedure, stimulation began before stretching and continued during the first part of shortening. In this case, the efficiency attained values of approximately 35% in sartorius, 50% in EDL, and 40% in soleus. These values are in rough agreement with those measured in vivo during running.
Actin synthesis rate and mRNA level increase during early recovery of atrophied muscle
Authors: Morrison P. R. , Muller G. W. , Booth F. W.
Published: December 2017

The purpose of this study was to correlate actin synthesis rate and alpha-actin mRNA level in the gastrocnemius-plantaris muscles of limbs during their recovery from 7 days of immobilization in 200- to 280-g female rats. The fractional synthesis rate of actin in control muscle was 1%/day. Actin synthesis rate was 33% of control level at the 7th day of hindlimb immobilization, returned to control value at the 2nd recovery day, and was three times higher than control on the 4th day of recovery. The alpha-actin mRNA was 53% of control at the 7th day of immobilization, and its increase during the 1st 2 recovery days paralleled the increase in actin synthesis rate; this suggests that pretranslational mechanisms caused the initial increase in actin synthesis. Further increases in actin synthesis from the 2nd to the 4th day appear to be under translational control, since actin synthesis was 300% of control on the 4th recovery day and alpha-actin mRNA was only 128% of control.
Hyperoxia, mitochondrial redox state, and lactate metabolism of in situ canine muscle
Authors: Wolfe B. R. , Graham T. E. , Barclay J. K.
Published: December 2017

The effect of hyperoxia on lactate production and release and the mitochondrial NAD+-to-NADH ratio was studied in the in situ canine gastrocnemius to determine whether elevated PO2 altered metabolic regulation. Dogs breathed either air (21% O2) [arterial O2 partial pressure (PaO2) 90 mmHg; n = 8] or hyperoxia (100% O2) (PaO2 546 mmHg; n = 8). The left muscle was stimulated for 10 min at 3 Hz and then both right and left muscles were quick frozen in N2. Hyperoxia did not affect O2 uptake, blood flow, and developed tension. Activity increased glucose 6-phosphate (G-6-P), D-fructose 6-phosphate (F-6-P), NH3, lactate, and F-6-P/F-1,6-P in both treatment groups. No significant differences in arterial or venous lactate, muscle lactate, glucose uptake, or glycogen depletion were noted in hyperoxia. Cytoplasmic NAD+/NADH was in a more oxidized state in hyperoxia at rest but not during activity. The increase in NH3 with stimulation was significantly larger in hyperoxia. Activity decreased alpha-ketoglutarate in hyperoxia but not in air. At stimulation, the estimated mitochondrial NAD+/NADH increased in both groups suggesting that hypoxia was not present. Thus hyperoxia did not affect mitochondrial redox state or lactate production and release in active muscle.
Changes in intracellular ionized Ca concentration associated with muscle fiber type transformation
Authors: Sreter F. A. , Lopez J. R. , Alamo L. , Mabuchi K. , Gergely J.
Published: December 2017

Since increased muscle activity, which results in fast-slow fiber transformation, is associated with increases in sarcoplasmic Ca2+ concentration ([Ca2+]i), it seemed of interest to study the level of [Ca2+] after cessation of stimulation in fibers of the extensor digitorum longus muscle chronically stimulated (8 Hz). [Ca2+]i was measured in individual fibers with a Ca2+-sensitive electrode after subtracting the membrane potential, measured simultaneously from the potential of the Ca2+ electrode. During the first 14 days of stimulation, [Ca2+]i increased from approximately 0.1 to 0.5 microM and declined in approximately 3 wk to a value slightly higher than the initial one. The rise and decline of [Ca2+]i was preceded by a transient increase in total calcium. If stimulation was terminated after 7-8 wk when an essentially complete fast-to-slow transformation had taken place, a subsequent rest period led to a reverse slow-to-fast transformation, which was also preceded by a transient increase of [Ca2+]i reaching a peak at day 5 of rest. Unstimulated fast and slow fibers and fully transformed fibers do not differ in their [Ca2+] levels; thus it appears that the transformation process itself is accompanied, particularly in its earlier stages, by elevated [Ca2+]i levels. Elucidation of the relation between changes in Ca2+ and changes in gene expression will require further work.