bluuebunny/crossref_metadata_2025 huggingface dataset:
Relaxation abnormalities in single cardiac myocytes from renovascular hypertensive rats
Authors: Yelamarty R. V. , Moore R. L. , Yu F. T. , Elensky M. , Semanchick A. M. , Cheung J. Y.
Published: December 2017

In myocardial hypertrophy secondary to renovascular hypertension, the rate of intracellular Ca2+ concentration decline during relaxation in paced left ventricular (LV) myocytes isolated from hypertensive (Hyp) rats is much slower compared with that from normotensive (Sham) rats. By use of a novel liquid-crystal television-based optical-digital processor capable of performing on-line real-time Fourier transformation and the striated pattern (similar to 1-dimensional diffraction grating) of cardiac muscle cells, sarcomere shortening and relaxation velocities were measured in single Hyp and Sham myocytes 18 h after isolation. There were no differences in resting sarcomere length, percent of maximal shortening, time to peak shortening, and average sarcomere shortening velocity between Sham and Hyp cardiac cells. In contrast, average sarcomere relaxation velocity and half-relaxation time were significantly prolonged in Hyp myocytes. Contractile differences between Sham and Hyp myocytes detected by the optical-digital processor are confirmed by an independent method of video tracking of whole cell length changes during excitation-contraction. Despite the fact that freshly isolated myocytes contract more rigorously than 18-h-old myocytes, the relaxation abnormality was still observed in freshly isolated Hyp myocytes, suggesting impaired relaxation is an intrinsic property of Hyp myocytes rather than changes brought about by short-term culture. We postulate that reduced sarcomere relaxation velocity is a direct consequence of impaired Ca2+ sequestration-extrusion during relaxation in Hyp myocytes and may be responsible for diastolic dysfunction in hypertensive hypertrophic myocardium at the cellular level.
Characterization of a delayed rectifier potassium current in chicken growth plate chondrocytes
Authors: Walsh K. B. , Cannon S. D. , Wuthier R. E.
Published: December 2017

With the use of the whole cell arrangement of the patch-clamp technique, an outward-directed time-dependent potassium current was identified in cultured chicken growth plate chondrocytes. This delayed rectifier potassium current (IK) activated with a sigmoidal time course during voltage steps to potentials positive to -40 mV. The half-maximal voltage required for current activation was determined to be -8 mV. The reversal potential (Erev) for IK, measured using deactivating tail currents, was -72 mV in the presence of 140 mM internal and 5 mM external [K+] solutions. Changes in external [K+] caused Erev to shift in a manner expected for a potassium-selective channel. In addition, increasing external [K+] from 5 to 50 mM caused the slope conductance of the tail currents to increase twofold. The chondrocyte IK was inhibited by the potassium-channel blocker 4-aminopyridine (4-AP) at concentrations of 0.5-4 mM and by the scorpion venom toxin charybdotoxin (CTX; 10 nM) but was unaffected by 10 mM tetraethylammonium (TEA). Addition of 20 microM ZnCl2 reduced IK in a voltage-dependent manner with the greatest inhibition found to occur at potentials near the threshold for current activation. Reduction of IK by ZnCl2 was accompanied by a slowing in the kinetics of IK activation. On the basis of the gating and pharmacological properties of this current, it is suggested that the chondrocyte channel belongs to a superfamily of K+ channels found in bone and immune system cells. The chondrocyte K+ channel may contribute to the unusually high [K+] found in the extracellular fluid of growth plate cartilage.
Norepinephrine-induced phosphatidylcholine hydrolysis by phospholipases D and C in rat tail artery
Authors: Gu H. , Trajkovic S. , LaBelle E. F.
Published: December 2017

Rat tail arterial segments were incubated with [3H]choline to selectively label endogenous phosphatidylcholine. Norepinephrine (NE; 10(-5) M) addition for periods of 10 s to 30 min significantly increased the concentration of extracellular phosphatidylcholine metabolites, [3H]choline, and [3H]phosphocholine. The release of [3H]choline and [3H]phosphocholine from the segments was NE dose dependent (10(-6)-10(-3) M). NE also increased the formation of [3H]phosphatidylethanol in [3H]myristate-labeled tail artery in the presence of ethanol, characteristic of phospholipase D activity. NE-induced phosphatidylcholine hydrolysis was blocked by pretreatment with prazosin (10(-5) M) and was unchanged by pretreatment with propranolol (10(-5) M). 4 beta-phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated the release of [3H]choline, which was inhibited by pretreatment with staurosporine (10(-5) M). The stimulatory effect of NE on phosphatidylcholine metabolism was not altered by either pretreatment with staurosporine (10(-5) M) or calcium-free buffer. In summary, we have demonstrated NE-stimulated phosphatidylcholine hydrolysis by phospholipase D and C in intact vascular smooth muscle. This effect of NE was dose dependent and was mediated through the alpha 1-adrenergic receptor. Norepinephrine and PDBu stimulated phosphatidylcholine hydrolysis through different mechanism(s), and the stimulatory effect of NE did not seem to require protein kinase C and calcium influx.
Contractile agonists activate voltage-dependent calcium channels in airway smooth muscle cells
Authors: Tomasic M. , Boyle J. P. , Worley J. F. , Kotlikoff M. I.
Published: December 2017

To determine whether agents that cause contraction of airway smooth muscle affect sarcolemmal calcium channel activity, unitary calcium channel currents (using Ba2+ as the charge carrier) were recorded in on-cell configuration from acutely dissociated (dog, pig, and ferret) and cultured (human) airway smooth muscle cells. Addition of the contractile agonists methacholine or bradykinin increased the open-state probability of the large-conductance calcium channel 37.2- and 45-fold, respectively. The increase in open-state probability was not due to cellular depolarization because increases occurred in the absence of depolarization. Channel activation by the agonist was determined to result in the favoring of a long (16.5 +/- 5.0 ms) open lifetime for the channel, which was not observed under control conditions, in the absence of BAY K 8644. We also report the unitary calcium channel currents from a second, smaller conductance calcium channel. This channel was present in all cell types and had a mean conductance of 9.5 +/- 0.8 pS (80 mM Ba2+). Exposure of cells to agonist also resulted in an increase in the open-channel probability of the small-conductance calcium channel (10.4-fold), which did not result from cellular depolarization. These experiments demonstrate that the molecular pathways exist between contractile agonist receptors and sarcolemmal calcium channels in airway smooth muscle cells. Because membrane patches were not directly exposed to agonist, receptor-channel linkage probably occurs via a second messenger-coupling pathway.
Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle
Authors: Vyas T. B. , Mooers S. U. , Narayan S. R. , Witherell J. C. , Siegman M. J. , Butler T. M.
Published: December 2017

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.
Outward currents in longitudinal colonic muscle cells contribute to spiking electrical behavior
Authors: Thornbury K. D. , Ward S. M. , Sanders K. M.
Published: December 2017

Electrical events in longitudinal and circular muscles of the colon are different. Longitudinal muscles generate action potentials superimposed upon small depolarizations termed myenteric potential oscillations and circular muscles generate slow wave events that persist for several seconds. Differences between circular and longitudinal muscles may be related to the potassium channels these cells express. We have studied Ca(2+)-dependent and voltage-dependent K currents of isolated longitudinal cells with the whole cell patch-clamp technique. Test depolarizations positive to -40 mV yielded a transient inward current followed by a large sustained outward current. Blockade of the inward Ca2+ current reduced the amplitude of the outward current. Outward current was also reduced by tetraethylammonium (TEA; 1 mM), suggesting that a component of the outward current is Ca2+ dependent. After blockade of the Ca(2+)-dependent outward current, a voltage- and time-dependent component of outward current remained. The activation and inactivation properties and sensitivity to TEA and 4-aminopyridine (4-AP) were characterized. The voltage-dependent outward current in longitudinal cells had different properties than the voltage-dependent K currents in circular muscle cells (i.e., more negative inactivation, less sensitivity to 4-AP). TEA (1-5 mM) increased the amplitude and frequency of action potentials in intact longitudinal muscles; 4-AP (1 mM) had little effect on electrical activity of longitudinal muscles. The data suggest that differences in electrical behavior of the 2 muscle layers may be related to the expression of different species of K channels.
Erythrocyte K-Cl cotransport: properties and regulation
Authors: Lauf P. K. , Bauer J. , Adragna N. C. , Fujise H. , Zade-Oppen A. M. , Ryu K. H. , Delpire E.
Published: December 2017

Erythrocytes possess a Cl-dependent, Na-independent K transport system cotransporting K and Cl in a 1:1 stoichiometry that is membrane potential independent. This K-Cl cotransporter is stimulated by cell swelling, acidification, Mg depletion, and thiol modification. Cell shrinkage, elevation of cellular divalent ions, thiol alkylation, phosphatase inhibitors, and derivatives of certain loop diuretics and stilbenes are inhibitory. Thus regulation of K-Cl cotransport at the membrane and cytoplasmic levels is highly complex. Basal K-Cl cotransport decreases with cellular maturation, whereas its modes of stimulation and inhibition are variable between species. The physiological inactivation appears to be prevented in low-K animal erythrocytes. In certain human hemoglobinopathies, K-Cl cotransport may be the cause of cellular dehydration and volume decrease. K-Cl cotransport occurs also in nonerythroid cells, such as in epithelial and liver cells of other species. At the threshold of molecular characterization, this comprehensive review places our present understanding of the mechanisms modulating K-Cl cotransport physiologically and pathophysiologically into kinetic and thermodynamic perspectives.
Potentiation of twitch contraction in guinea pig ureter by sodium vanadate
Authors: Sunano S. , Moriyama K. , Shimamura K.
Published: December 2017

The effect of sodium vanadate on action potential and twitch contraction of guinea pig ureter was studied and compared with that of ouabain, elevated K+, low temperature, and a Ca agonist (BAY K 8644), which can be expected to exert certain comparative effects. Sodium vanadate markedly potentiated twitch contraction. Potentiation by vanadate was associated with marked prolongation of relaxation time. Sodium vanadate caused only slight depolarization of the membrane but marked changes in action potential. The duration of action potential was prolonged and the number of oscillatory spike potentials increased. These effects were different from those of other treatments. It is concluded that prolongation of action potential and the increase in the number of spikes are the main cause of potentiation of twitch contraction by sodium vanadate. In addition, inhibition of Ca pump activity of the smooth muscle membrane system by vanadate might also be involved in potentiation of twitch contraction.
PGE2 regulates cAMP production in cultured rabbit CCD cells: evidence for dual inhibitory mechanisms
Authors: Noland T. D. , Carter C. E. , Jacobson H. R. , Breyer M. D.
Published: December 2017

In cultured cortical collecting duct (CCD) cells, exogenous prostaglandin E2 (PGE2) inhibited arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in a concentration-dependent manner. Although pertussis toxin (PT, 500 ng/ml) alone did not reverse the PGE2-dependent inhibition, PT and staurosporine, a protein kinase C inhibitor, together partially reversed the effect of exogenous PGE2. In contrast, PT completely reversed the inhibition of AVP-dependent cAMP production by sulprostone. These data suggest that exogenous PGE2 can inhibit AVP-stimulated cAMP production and that the inhibitory effects of PGE2 are mediated by staurosporine- and PT-sensitive component(s). Short-term (15-240 min) incubation with phorbol 12-myristate 13-acetate (PMA, 10(-7) M) inhibited PGE2-stimulated cAMP production. Long-term (20 h) incubation with PMA augmented PGE2-stimulated cAMP production. These data provide evidence for the maintenance of a PT-sensitive PGE2-dependent inhibitory pathway of cAMP production in cultured CCD cells. In addition, data are presented that support an inhibitory role for protein kinase C in the effects of PGE2 on the metabolism of cAMP in these cells.
Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ
Authors: Vasseur M. , Frangne R. , Alvarado F.
Published: December 2017

The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the modulation of SPQ fluorescence by commonly used buffers was investigated as a function of pH. Titration curves demonstrated SPQ quenching and yielded pK values characteristic of each buffer. from here, pH-independent Stern-Volmer constants, KQbase, were calculated for each of the morpholine derivatives, MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine derivatives, exhibited an additional pH-independent "molecular" quenching constant KmQ throughout the pH range 3-10. To study chloride fluxes, therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl, which is a function of both pH and buffer composition. Equations describing these relationships are presented. In conclusion, unless both pH and the buffer composition are taken into account, SPQ is unsuitable for studying the concomitant transmembrane fluxes of Cl- and H+.
Mechanisms of adaptation of glucose transporters to changes in the oxidative chain of muscle and fat cells
Authors: Bashan N. , Burdett E. , Guma A. , Sargeant R. , Tumiati L. , Liu Z. , Klip A.
Published: December 2017

Cells in which glucose uptake is rate limiting respond to hypoxic insults with an increase in glucose transport activity. To understand the underlying cellular mechanisms involved in this adaptive response, the effects of an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol (DNP), and of an inhibitor of the electron transport chain, rotenone, were compared with the effect of hypoxia in L6 muscle cells and 3T3-L1 adipocytes. All three conditions (DNP, rotenone, and 3% oxygen) elevated hexose uptake by approximately twofold in 4 h relative to control cells. All three insults decreased cellular ATP levels rapidly. A subsequent recovery was observed within 1-2 h in the presence of DNP or 3% oxygen, probably as a result of anaerobic production of ATP through increased glucose uptake and glycolysis. DNP and rotenone elevated the content of GLUT-1 protein in isolated plasma membranes and decreased it in intracellular light microsomes, suggestive of translocation of this transporter isoform. No change in GLUT-4 protein distribution was detected. In contrast, 3% oxygen caused a marked specific increase in GLUT-1 protein in both plasma membranes and microsomes. Consistently, cycloheximide had no effect on the hexose transport responses to DNP or rotenone, but prevented the response to hypoxia. However, GLUT-1 mRNA and the total cell content of GLUT-1 protein were elevated by all three treatments. It is proposed that within the time frame studied, reductions in the energy charge may activate the glucose transport system in L6 myotubes and 3T3-L1 adipocytes by GLUT-1 protein biosynthesis and translocation. When both responses exist, the biosynthetic pathway is dispensable, and posttranslational mechanisms, including transporter translocation suffice to sustain the adaptive elevation in glucose transport activity for several hours. s
Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes
Authors: Hove-Madsen L. , Bers D. M.
Published: December 2017

Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 microM thapsigargin and 30 microM ruthenium red to prevent sarcoplasmic reticulum (SR) or mitochondrial Ca uptake. Passive Ca binding was obtained by titration of the myocytes with Ca and subtraction of Ca binding in a blank titration without myocytes. Passive Ca binding could be described by a Michaelis binding curve with two sites: K1 = 0.42 microM n1 = 1.27 nmol/mg cell protein and K2 = 79 microM, n2 = 4.13 nmol/mg cell protein. The passive Ca buffering over the physiological Ca concentration was approximately twice the value expected from the values compiled by Fabiato [A. Fabiato. Am. J. Physiol. 245 (Cell Physiol. 14): C1-C14, 1983]. The maximal SR Ca uptake in the presence of 30 microM ruthenium red was fit by an uptake curve with a maximal uptake of 5.16 nmol/mg cell protein and a K 1/2 of 1.0 microM. In the presence of 5 microM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 microM free Ca. Rapid changes in free Ca concentration ([Ca]) measured with a Ca electrode were slower than simultaneous measurements of free [Ca] with indo-1 in permeabilized myocytes. However, oxalate, which buffers Ca and maximizes SR Ca uptake, increased the uptake rate and eliminated the difference in free [Ca] measured with Ca electrode and indo-1. This suggests that spatial gradients of [Ca] exist in permeabilized myocytes without Ca buffering. The new estimates of the buffering of intracellular Ca in cardiac myocytes should be valuable in developing quantitative insights into cardiac Ca regulation.
Modulation of cytokines and myocardial lesions by vitamin E and chloroquine in a Mg-deficient rat model
Authors: Weglicki W. B. , Stafford R. E. , Freedman A. M. , Cassidy M. M. , Phillips T. M.
Published: December 2017

Antioxidant treatment with alpha-tocopherol did not affect the level of the "inflammatory cytokines" in Mg-deficient animals, although it diminished the extent of the myocardial lesions. In another group of Mg-deficient animals chloroquine treatment diminished significantly the levels of circulating cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) and also resulted in a major decrease in myocardial lesions. These results raise the question of the role of these inflammatory cytokines in the formation of lesions in Mg-deficient myocardium. Because these cytokines are able to stimulate free radical production in various cell types, we postulate that Mg deficiency involves free radical mechanisms that can be amplified by inflammatory cytokines; whether these cytokines initiate lesion formation is unclear. Although our data do not confirm either possibility, we submit that these results implicate a role for the inflammatory cytokines in the cardiac pathology of Mg deficiency.
Sorting and recycling efficiency of apical insulin binding sites during endocytosis in proximal tubule cells
Authors: Nielsen S.
Published: December 2017

Intracellular traffic and recycling of apical insulin binding sites are examined in isolated, perfused proximal tubules. The endocytic binding sites were specific as revealed by 90% reduction in 125I-labeled insulin binding by 10(-5) M insulin. The traffic was followed by developing a chemical cross-linking method to covalently label the binding sites. Only 3% of cross-linked insulin-gold was transported to the lysosomes, reflecting high sorting and recycling efficiency. Correspondingly, only 4% of cross-linked 125I-insulin was degraded, and only 5% of the electron microscopy-autoradiographic grains was associated with lysosomes. No label was transferred to the Golgi apparatus; thus neither lysosomes nor Golgi apparatus is involved in the recycling. In contrast, approximately 40% of non-cross-linked ligand was transferred to the lysosomes. Tubules first pretreated with cross-linker and then perfused with insulin-gold or 125I-labeled insulin-like growth factor I revealed lysosomal accumulation and degradation at control levels. Thus the cross-linker does not interfere with membrane or protein processing. The study also provides evidence for a vesicular transtubular transport because insulin-gold was transcytosed to the basolateral part of the cells and to the intracellular spaces (0.5%). In contrast cross-linked label was never observed in intercellular spaces, suggesting sorting of apical binding sites, a mechanism contributing to maintenance of cell polarity. In conclusion, traffic, sorting, and recycling of binding sites take place with high efficiency.
Stimulation of prostaglandin synthesis by human endothelial cells exposed to hypoxia
Authors: Michiels C. , Arnould T. , Knott I. , Dieu M. , Remacle J.
Published: December 2017

In ischemic organs, arachidonic acid (AA) metabolites and mostly prostaglandins (PGs) have been found to be released in high amounts. The mechanism for this AA metabolism activation and its physiological implications are not clear. Because endothelial cells are an important source of PGs and because they seem to be very rapidly affected by ischemia, we developed an in vitro model where human endothelial cells were submitted to hypoxia. An important specific activation of phospholipase A2 was observed during hypoxia, which was concomitant with a rise in cytosolic calcium concentration. Endothelial cells synthetize in normal conditions as a mean 1.42, 1.00, 7.69, and 26.92 ng/mg proteins of, respectively, PGE2, PGD2, PGF2 alpha, PGI2. An important increase of about five- to ninefold in the synthesis of the four PGs was observed during hypoxia, which followed the same kinetics as the PLA2 activation. This increase in PG synthesis was sensitive to cyclooxygenase inhibitors. During reoxygenation, PG synthesis decreased back to the basal level of resting cells, suggesting that cells were able to recover their homeostasis after hypoxia. These observations indicate that endothelial cells exposed to oxygen deprivation are a major source of PGs and could contribute to the high amounts of PG released in vivo in ischemic organs.
Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes
Authors: Dawson T. L. , Gores G. J. , Nieminen A. L. , Herman B. , Lemasters J. J.
Published: December 2017

Cell killing, oxygen consumption, and hydroperoxide formation were determined in rat hepatocytes after glycolytic and respiratory inhibition. These conditions model the ATP depletion and reductive stress of anoxia (“chemical hypoxia”). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cyanide, or myxothiazol. Cell killing, hydroperoxide formation, and inhibitor-insensitive oxygen consumption were greater after azide than after myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation, delayed cell killing after each of the respiratory inhibitors. Anoxia also delayed cell killing during chemical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reactive oxygen species participate in lethal cell injury during chemical hypoxia. In isolated mitochondria, previous studies have shown that myxothiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone can react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involved in cell killing during reductive stress. In support of this hypothesis, myxothiazol reduced rates of cell killing and hydroperoxide formation in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative but not absolute hypoxia.
Purine nucleoside formation in rat skeletal muscle fiber types
Authors: Arabadjis P. G. , Tullson P. C. , Terjung R. L.
Published: December 2017

To determine the capacity for purine nucleotide degradation among skeletal muscle fiber types, we established energy-depleted conditions in muscles of the rat hindlimb by inducing muscle contraction during ischemia. After 5, 10, 15, or 20 min of ischemic contractions, representative muscle sections were freeze-clamped and analyzed for purine nucleotides, nucleosides, and bases. Fast-twitch muscle sections accumulated about fourfold more IMP than the slow-twitch red soleus muscle. Inosine begins to accumulate at < 0.5 mumol/g IMP in slow-twitch muscle and at approximately 2 mumol/g IMP in fast-twitch muscle. This suggests that inosine is formed intracellularly by 5'-nucleotidase acting on IMP and that the activity and/or substrate affinity of the 5'-nucleotidase present in slow-twitch muscle may be higher than in fast-twitch muscle. At similar concentrations of precursor IMP, slow-twitch muscle has a greater capacity for purine nucleoside formation and should be more dependent on salvage and de novo synthesis of purine for the maintenance of muscle adenine nucleotides. Fast-twitch muscles are better able to retain IMP for subsequent reamination due to their lower capacity to degrade IMP to inosine.
Role of Na-K-Cl cotransport in vascular endothelial cell volume regulation
Authors: O'Donnell M. E.
Published: December 2017

Vascular endothelial cells have been shown previously to possess a highly active Na-K-Cl cotransport system that mediates the major portion of total K influx and is regulated by a variety of vasoactive hormones and neurotransmitters. These observations suggest that the cotransporter may be an important component of endothelial cell function. The present study was conducted to investigate the role of Na-K-Cl cotransport in regulation of endothelial cell volume. Cultured bovine aortic endothelial cells were exposed to media of varying tonicities and Na-K-Cl cotransport activity assessed as bumetanide-sensitive K influx. Increasing the extracellular tonicity by increments as small as 10 mosM was found to cause significant stimulation of cotransport activity, and lowering tonicity reduced activity of the transporter. Exposure of endothelial cells to hypertonic medium was also found to increase bumetanide-sensitive net uptake of Na and K and total cellular Na and K content. Endothelial cell volume was evaluated by [14C]urea determination of intracellular water space in endothelial monolayers and by electronic cell sizing of suspended cells. Treatment of the cells with agents that stimulate Na-K-Cl cotransport activity was found to increase cell volume, whereas cotransport-inhibiting agents decreased cell volume. Exposure of the cells to hypertonic medium caused a rapid decrease in cell volume, followed by a regulatory volume increase that was greatly attenuated by bumetanide. The volume recovery was partially inhibited by the Na-H exchange inhibitor amiloride and was nearly abolished by bumetanide and amiloride in combination. Endothelial cells of pulmonary artery and cerebral microvessels were also found to exhibit increased Na-K-Cl cotransport activity on exposure to hypertonic media. These findings suggest that Na-K-Cl cotransport is of major importance in endothelial cell volume regulation.
G protein coupling of human platelet V1 vascular vasopressin receptors
Authors: Thibonnier M. , Goraya T. , Berti-Mattera L.
Published: December 2017

We used several approaches to identify the G protein coupled to V1 vascular arginine vasopressin (AVP) receptors of human platelets. In purified platelet membranes, high-affinity specific binding of [3H]AVP but not that of the V1 vascular antagonist [3H]d(CH2)5Tyr(Me)AVP was modulated by guanosine 5'-O-(3-thiotriphosphate) or sodium fluoride both in the presence and absence of MgCl2. AVP failed to modify the [alpha-32P]GTP labeling pattern or the cytosolic translocation of the 24- to 27-kDa GTP-binding proteins. AVP-stimulated GTPase activity of platelet membranes was blocked by antibodies specific for the COOH-terminal of the Gq alpha protein. AVP increased labeling of a 42-kDa platelet membrane protein by the photoreactive GTP analogue [alpha-32P]azidoanilido GTP. Immunoblotting of platelet proteins with various G protein-specific antibodies revealed that the 42-kDa protein labeled with [alpha-32P]azidoanilido GTP was immunoblotted only by antibodies specific for the alpha-subunit of GQ-11. Thus V1 vascular AVP receptors of human platelets are coupled in a divalent cation-dependent manner to a G protein belonging to the Gq-11 family.
Modulation by extracellular ATP of two distinct currents in rat myocytes
Authors: Zheng J. S. , Christie A. , Levy M. N. , Scarpa A.
Published: December 2017

The whole cell voltage-clamp technique was used to study the electrophysiological effects induced by ATP in isolated ventricular myocytes. ATP or 2-methylthio-ATP evoked a transient inward current (IATP) when the transmembrane potential (Vm) was held at -70 mV and increased the Ca2+ current (ICa) when Vm was depolarized to 0 mV. The time course of IATP was fitted by a single exponential equation with a brief time constant (165 ms), whereas the time course of enhancement of ICa by ATP was also fitted by a single exponential equation with a much longer time constant (14 s). IATP was much less pronounced when extracellular Mg2+ was absent, and it was insensitive to dihydropyridines. In contrast, the enhancement of ICa by ATP was not affected by removing extracellular Mg2+, but it was suppressed by Ca2+ channel blockers. Both IATP and ICa were decreased by extracellular Cd2+. Internally applied guanosine 5'-O-(2-thiodiphosphate), which prevents the activation of G proteins, abolished the ATP-enhanced rise in ICa but did not inhibit IATP. These data suggest that ATP elicits IATP and increases ICa through two different mechanisms. IATP appears to be generated via receptor-operated channels that are activated by ATP. The ATP-induced increase of ICa appears to be mediated by G proteins via pathways that are independent of adenosine 3',5'-cyclic monophosphate and phosphoinositide turnover.
Increases in human skeletal muscle Na(+)-K(+)-ATPase concentration with short-term training
Authors: Green H. J. , Chin E. R. , Ball-Burnett M. , Ranney D.
Published: December 2017

To investigate the effect of short-term training on Na(+)-K(+)-adenosine triphosphatase (ATPase) concentration in skeletal muscle and on plasma K+ homeostasis during exercise, 9 subjects performed cycle exercise for 2 h per day for 6 consecutive days at 65% of maximal aerobic power (VO2 max). Na(+)-K(+)-ATPase concentration determined from biopsies obtained from the vastus lateralis muscle using the [3H]ouabain-binding technique increased 13.6% (P < 0.05) as a result of the training (339 +/- 16 vs. 385 +/- 19 pmol/g wet wt, means +/- SE). Increases in Na(+)-K(+)-ATPase concentration were accompanied by a small but significant increase in VO2 max (3.36 +/- 0.16 vs. 3.58 +/- 0.13 l/min). The increase in arterialized plasma K+ concentration and plasma K+ content determined during continuous exercise at three different intensities (60, 79, and 94% VO2 max) was depressed (P < 0.05) following training. These results indicate that not only is training capable of inducing an upregulation in sarcolemmal Na(+)-K(+)-ATPase concentration in humans, but provided that the exercise is of sufficient intensity and duration, the upregulation can occur within the first week of training. Moreover, our findings are consistent with the notion that the increase in Na(+)-K(+)-ATPase pump concentration attenuates the loss of K+ from the working muscle.
Ratio of ryanodine to dihydropyridine receptors in cardiac and skeletal muscle and implications for E-C coupling
Authors: Bers D. M. , Stiffel V. M.
Published: December 2017

We measured dihydropyridine receptor (DHPR) and ryanodine receptor (RYR) density in isolated ventricular myocytes from rabbits, rats, ferrets, and guinea pigs and also from rabbit ventricular homogenate, skeletal muscle homogenate, and isolated triads. In skeletal muscle homogenate and triads the RYR/DHPR ratio was 0.7 and 0.52, respectively. This stoichiometry is reasonably consistent with excitation-contraction (E-C) coupling models in skeletal muscle where the DHPR molecule itself may transmit the signal for Ca release to the sarcoplasmic reticulum (SR) and with the molecular arrangement proposed for toadfish swimbladder from ultrastructural studies by B. A. Block, T. Imagawa, K. P. Campbell, and C. Franzini-Armstrong. (J. Cell Biol. 107: 2587-2600, 1988). That is, there could be approximately two RYR for each four DHPR or two RYR feet per DHPR tetrad in an organized array (assuming 1 high-affinity RYR/foot and 4 DHPR/tetrad). The fraction of rabbit ventricular protein that is cardiac myocyte protein was also estimated (< or = 55-62%), assuming that RYR and DHPR are useful but not exclusive markers for myocytes in the ventricle. In cardiac myocytes the RYR/DHPR was much higher than in skeletal muscle and varied among different mammalian myocytes. The RYR/DHPR ratios were 3.7 in rabbit, 4.3 in guinea pig, 7.3 in rat, and 10.2 in ferret myocytes. In contrast to skeletal muscle, these results indicate that there are many more RYR feet per DHPR in cardiac muscle, and this ratio depends on species (i.e., 4-10 times and would be 4 times higher still per putative DHPR tetrad if that structure exists in mammalian heart).(ABSTRACT TRUNCATED AT 250 WORDS)
Evaluation of antitumor effect of tumor necrosis factor in terms of protein metabolism and cell cycle kinetics
Authors: Wan J. M. , Fogt F. , Bistrian B. R. , Istfan N. W.
Published: December 2017

To determine the significance of protein breakdown in regulating tumor growth and to better understand the antitumor mechanism of tumor necrosis factor in vivo, we measured the effects of a 6-h constant intravenous infusion of human recombinant tumor necrosis factor-alpha (rHuTNF) on tumor protein metabolism and cell cycle kinetics in rats bearing the Walker-256 carcinosarcoma. Protein metabolism was investigated with the use of [14C]leucine infusion; estimates of tumor cell cycle kinetics were obtained in vivo by use of 5-bromo-2'-deoxyuridine (BrdUrd) pulse labeling and bivariate BrdUrd/DNA analysis by flow cytometry. Reduction in tumor growth by rHuTNF was associated with a dose-dependent increase in tumor proteolysis but no change in tumor protein synthesis. At the cellular level, rHuTNF had a significant cytostatic effect on G2/M cells and caused a marked decrease in the fraction of cells capable of BrdUrd uptake. Release of BrdUrd, an indicator of cell death, was noted in only 7.5% of tumor cells labeled at the beginning of rHuTNF infusion. These results suggest that either tumor protein breakdown may influence cell cycle activity by regulating cytoplasmic protein mass or that tumor proteolysis may be a compensatory mechanism for limiting cytoplasmic size when cellular division is interrupted suddenly.
Expression of the epithelial Na+ channel in the developing rat lung
Authors: O'Brodovich H. , Canessa C. , Ueda J. , Rafii B. , Rossier B. C. , Edelson J.
Published: December 2017

The adult mature fetal, but not immature fetal, lung is capable of actively transporting Na+ from the alveolar space. The reason for the impaired Na+ transport in the immature lung is not known; however, the apical membrane Na+ channel is the rate-limiting step for epithelial Na+ transport. This study determined whether transcripts coding for the adult rat colonic epithelial Na+ channel (alpha rENaC) were present in the fetal and adult lung and whether they were developmentally regulated. Similarly sized alpha rENaC transcripts were identified in RNA isolated from fetal and adult whole rat lung, primary cultures of fetal and adult alveolar epithelium, and adult rat whole kidneys, suggesting that the lung alpha rENaC is a similar transcript to that found in the salt-deprived rat colonic epithelium. There were low mRNA levels in 17- to 18-day gestational age (GA) fetal lungs and epithelium (term GA = 22 days), but these levels increased markedly during the saccular stage of lung development (20 days GA) and remained high in adult lungs. The combined administration of thyroid-releasing hormone and dexamethasone to pregnant rats between 16 and 18 days GA induced the expression of lung alpha rENaC in their fetuses. We conclude that alpha rENaC is expressed in mature fetal and adult alveolar epithelium and that it is influenced by hormones known to alter maturation of the fetal lung.
Adult human aortic smooth muscle cells in culture produce active TGF-beta
Authors: Kirschenlohr H. L. , Metcalfe J. C. , Weissberg P. L. , Grainger D. J.
Published: December 2017

Vascular smooth muscle cells (VSMC) from adult human aortas proliferated in culture in response to fetal calf serum (FCS) with a population doubling time of 70-85 h compared with 35 +/- 5 h for VSMC derived from adult rat aortas. Medium conditioned on cultures prepared from aortas from three different donors and mixed 1:1 with fresh Dulbecco's modified Eagle's medium plus 20% FCS [human conditioned medium (HCM)] reduced the rate of proliferation of rat VSMC by 46 +/- 6% (n = 3) after 48 h compared with cells in fresh medium. HCM did not reduce the proportion (> 65%) of rat VSMC that entered DNA synthesis but delayed entry into mitosis by at least 18 h. This effect was similar to previous observations of the action of transforming growth factor-beta (TGF-beta) on rat VSMC (G. K. Owens, A. A. Geisterfer, Y. W. Yang, and A. Komoriya. J. Cell Biol. 107: 771-780, 1988). A TGF-beta assay using DNA synthesis in mink lung epithelial cells confirmed that human, but not rat, VSMC in culture secrete active TGF-beta. Addition of a neutralizing antibody to TGF-beta to human VSMC in the presence of 20% FCS decreased the population doubling time from 74 +/- 3 to 46 +/- 6 h (n = 3). These observations demonstrate that the long population doubling time of human VSMC is due to the production of active TGF-beta and to an inhibitory autocrine loop.
Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies
Authors: Porzig H. , Li Z. , Nicoll D. A. , Philipson K. D.
Published: December 2017

We used a panel of monoclonal antibodies raised against the canine cardiac Na(+)-Ca2+ exchanger expressed in Sf9 insect cells to analyze the immunoreactive domains and the topological organization of this membrane protein. Antibodies, which reacted strongly on Western blots of the recombinant protein, were used to screen an expression sublibrary composed of exchanger cDNA fragments. Positive clones thus indicated the expression of antibody binding sites. Linear epitopes, 16-155 amino acids in length, could be identified for four antibodies. One antibody recognized two neighboring, but nonoverlapping, sequences. All epitopes were localized to the large hydrophilic region of the exchanger connecting the putative transmembrane segments 5 and 6. The immunodominant region of the protein is a highly charged domain in the carboxy-terminal half of the hydrophilic region. Binding studies with the 3H-labeled high-affinity antibody R3F1 establish that the immunodominant region is located on the intracellular surface of the membrane. The same antibody was used to directly determine the membrane concentration of the exchanger in different cell types. Newborn rat heart cells contain approximately 6 x 10(5) exchanger molecules per cell. Exchanger densities in different cells seem to correlate with the Na(+)-dependent Ca2+ transport activity in the corresponding membrane vesicles.
Adaptations in myosin heavy chain expression and contractile function in dystrophic mouse diaphragm
Authors: Petrof B. J. , Stedman H. H. , Shrager J. B. , Eby J. , Sweeney H. L. , Kelly A. M.
Published: December 2017

The X chromosome-linked muscular dystrophic (mdx) mouse lacks the subsarcolemmal protein dystrophin and thus represents a genetic homologue of human Duchenne muscular dystrophy. The present study examined alterations in diaphragm contractile properties and myosin heavy chain (MHC) expression in young (3-4 mo) and old (22-24 mo) control and mdx mice. In young mdx mice, maximum isometric tension (Po) was reduced to 50% of control values. An increase in fibers coexpressing types I (slow) and IIa MHC as well as regenerating fibers expressing embryonic MHC occurred, whereas IIx/b fibers were decreased. In the old mdx group, Po underwent a further reduction to 25% of control, and there was a slowing of twitch kinetics along with markedly increased diaphragm endurance. These changes were associated with an approximate sevenfold increase in type I MHC fibers and virtual elimination of the IIx/b fiber population; there was no detectable embryonic MHC expression. We conclude that the mdx diaphragm responds to progressive muscle degeneration with transition to a slower phenotype associated with reduced power output and augmented muscle endurance. In the setting of progressive muscle fiber destruction, these changes may help preserve contractile function and promote greater survival of remaining muscle fibers by decreasing cellular energy requirements.
Temporal patterns of alpha 1-receptor stimulation regulate amplitude and frequency of calcium transients
Authors: Schofl C. , Brabant G. , Hesch R. D. , von zur Muhlen A. , Cobbold P. H. , Cuthbertson K. S.
Published: December 2017

The pulsatile release of neurotransmitters and many hormones might encode specific biological information according to temporal pattern. We tested this hypothesis by applying pulsed alpha 1-adrenoceptor stimulation to single aequorin-injected hepatocytes. The amplitude of free Ca2+ transients induced by rapid phenylephrine pulses (20-s interpulse interval) and continuous stimulation was similar (approximately 640 nM) but increased to approximately 1,000 nM as the interpulse interval was increased to 120 s. The same overall response was maintained despite a 13-fold reduction in average phenylephrine concentration. Some regimes of pulsed phenylephrine stimulation could give a higher frequency of pulsed phenylephrine stimulation could give a higher frequency of free calcium oscillations than continuous stimulation, or more rapid stimulation when some agonist pulses failed to elicit a free Ca2+ transient. For the same average phenylephrine concentration (0.3-0.6 microM), pulsed regimes could result in significantly higher frequencies and integrated responses than constant application. The lags between phenylephrine pulses and free Ca2+ transients reduced as the period between pulses increased. The amplitude and lag data are consistent with a refractory period of 18 s and a recovery phase with a time constant of approximately 100 s, perhaps corresponding to dephosphorylation of alpha 1-adrenoceptors phosphorylated by protein kinase C during each free Ca2+ transient.
Cell volume analysis of gramicidin-treated eccrine clear cells to study regulation of Cl channels
Authors: Ohtsuyama M. , Suzuki Y. , Samman G. , Sato F. , Sato K.
Published: December 2017

Using voltage-current-clamp methods, we determined membrane potentials, relative ionic permeability, and membrane conductance of gramicidin (GC)-treated freshly dissociated eccrine clear cells. GC depolarized the membrane potential by 58 mV, increased the membrane conductance progressively over the time of exposure (mean of 1.7 times at 60 s and 4.6 times at 3 min), and increased the Na conductance of the membrane (from near 0 in control to 0.75 nS after GC). Image analysis coupled with GC treatment was then employed to study the regulation of Cl channels based on the premise that cell swelling was due to activation of Cl channels. Cell swelling was stimulated by methacholine (MCh, 3 microM) in the presence of GC. GC+MCh-induced cell swelling was inhibited by atropine, low extracellular Ca ([Ca]o < 1 nM), or removal of Cl. Thus MCh-induced cell swelling is most likely due to Ca-dependent activation of Cl channels. Isoproterenol (Iso), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, and forskolin also caused cell swelling in the presence of GC. Iso-induced cell swelling was abolished in a Cl-free medium and by diphenylamine-2-carboxylic acid, indicating that it is caused by adenosine 3',5'-cyclic monophosphate (cAMP)-mediated activation of Cl channels. Cl channels stimulated by MCh, but not those stimulated by Iso, were inhibited by preexposure to a low-Ca medium [nominally Ca free + 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, [Ca]o < 1 nM] for 20 s, suggesting that Ca-stimulated Cl channels are distinct from cAMP-dependent Cl channels. cAMP-stimulated Cl channels were, however, inhibited when the cells were exposed to the low-Ca medium for 60 s. The simple cell volume analysis of GC-treated cells is a sensitive assay system for both Ca- and cAMP-dependent Cl channels.(ABSTRACT TRUNCATED AT 250 WORDS)