scholarly journals The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter

2018 ◽  
Vol 19 (9) ◽  
pp. 2489 ◽  
Author(s):  
Lin Zhang ◽  
Christian Aalkjaer ◽  
Vladimir Matchkov
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Isometric Force ◽  
Src Kinase ◽  
Ouabain Binding ◽  
Arterial Diameter ◽  
Small Arteries ◽  
Functional Changes ◽  
High Affinity ◽  
Different Diameters

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150–350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.

Myosin thick filament lability induced by mechanical strain in airway smooth muscle

2001 ◽  
Vol 90 (5) ◽  
pp. 1811-1816 ◽  
Author(s):  
Kuo-Hsing Kuo ◽  
Lu Wang ◽  
Peter D. Paré ◽  
Lincoln E. Ford ◽  
Chun Y. Seow
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Cross Sections ◽  
Structural Changes ◽  
Isometric Force ◽  
Mechanical Strain ◽  
Thick Filament ◽  
Functional Changes ◽  
Thick Filaments ◽  
Length Changes

Airway smooth muscle adapts to different lengths with functional changes that suggest plastic alterations in the filament lattice. To look for structural changes that might be associated with this plasticity, we studied the relationship between isometric force generation and myosin thick filament density in cell cross sections, measured by electron microscope, after length oscillations applied to the relaxed porcine trachealis muscle. Muscles were stimulated regularly for 12 s every 5 min. Between two stimulations, the muscles were submitted to repeated passive ±30% length changes. This caused tetanic force and thick-filament density to fall by 21 and 27%, respectively. However, in subsequent tetani, both force and filament density recovered to preoscillation levels. These findings indicate that thick filaments in airway smooth muscle are labile, depolymerization of the myosin filaments can be induced by mechanical strain, and repolymerization of the thick filaments underlies force recovery after the oscillation. This thick-filament lability would greatly facilitate plastic changes of lattice length and explain why airway smooth muscle is able to function over a large length range.

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

scholarly journals Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study

1994 ◽  
Vol 297 (2) ◽  
pp. 415-423 ◽  
Author(s):  
M Wibo ◽  
T Godfraind
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Gradient Centrifugation ◽  
Ryanodine Receptors ◽  
Intestinal Smooth Muscle ◽  
Affinity Binding ◽  
Stoichiometric Ratio ◽  
Cell Fractionation ◽  
High Affinity Binding ◽  
High Affinity

[3H]Ins(1,4,5)P3- and [3H]ryanodine-binding sites were characterized in membrane fractions from guinea-pig intestinal smooth muscle (longitudinal layer) and their subcellular localization was investigated by analytical cell-fractionation techniques. Fractions collected at low centrifugal fields (N and M fractions) contained predominantly low-affinity [3H]Ins(1,4,5)P3-binding sites (KD 80 nM), whereas microsomal (P) fractions contained only high-affinity binding sites (KD 5 nM). Total sedimentable high-affinity binding sites of [3H]Ins(1,4,5)P3 were 9-10-fold more numerous than those of [3H]ryanodine. Both high-affinity binding sites were purified in microsomal fractions, and their sub-microsomal distribution patterns after isopycnic density-gradient centrifugation were similar to those of presumed endoplasmic reticulum (ER) constituents, indicating that Ins(1,4,5)P3 and ryanodine receptors were localized primarily in ER and probably associated with rough as well as smooth ER. However, the stoichiometric ratio of Ins(1,4,5)P3 to ryanodine receptors was distinctly higher in high-density RNA-rich subfractions than in low-density RNA-poor subfractions, suggesting that Ins(1,4,5)P3 receptors were somewhat concentrated in the ribosome-coated portions of ER. The low overall stoichiometric ratio of ryanodine to Ins(1,4,5)P3 receptors in intestinal smooth muscle (1:9-10) might explain, at least partly, the existence of a Ca(2+)-storage compartment devoid of ryanodine-sensitive Ca2+ channels, but equipped with Ins(1,4,5)P3-sensitive channels, in saponin-permeabilized smooth-muscle cells [Iino, Kobayashi and Endo (1988) Biochem. Biophys. Res. Commun. 152, 417-422].

Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat

1989 ◽  
Vol 490 (1) ◽  
pp. 170-173 ◽  
Author(s):  
Sofia I. Pylova ◽  
Joanna Majkowska ◽  
Wojciech Hilgier ◽  
Andrzej Kapuścinśki ◽  
Jan Albrecht
Keyword(s):  
Cerebral Ischemia ◽  
Binding Sites ◽  
Rapid Decrease ◽  
Global Cerebral Ischemia ◽  
Ouabain Binding ◽  
Short Term ◽  
High Affinity ◽  
Ca1 Region ◽  
Hippocampal Ca1 Region ◽  
Hippocampal Ca1

Skeletal muscle ouabain binding sites are reduced in rats with chronic heart failure

2002 ◽  
Vol 92 (6) ◽  
pp. 2326-2334 ◽  
Author(s):  
Timothy I. Musch ◽  
Swen Wolfram ◽  
K. Sue Hageman ◽  
Joel G. Pickar
Keyword(s):  
Heart Failure ◽  
Skeletal Muscle ◽  
Chronic Heart Failure ◽  
Oxygen Uptake ◽  
Binding Sites ◽  
Ouabain Binding ◽  
High Affinity ◽  
Lv Dysfunction ◽  
Run Time ◽  
Fast Twitch

Intrinsic skeletal muscle abnormalities decrease muscular endurance in chronic heart failure (CHF). In CHF patients, the number of skeletal muscle Na+-K+ pumps that have a high affinity for ouabain (i.e., the concentration of [3H]ouabain binding sites) is reduced, and this reduction is correlated with peak oxygen uptake. The present investigation determined whether the concentration of skeletal muscle [3H]ouabain binding sites found during CHF is related to 1) severity of the disease state, 2) muscle fiber type composition, and/or 3) endurance capacity. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types. Measurements were obtained 8–10 wk postsurgery in 23 myocardial infarcted (MI) and 18 sham-operated control (sham) rats. Eighteen rats had moderate left ventricular (LV) dysfunction [LV end-diastolic pressure (LVEDP) < 20 mmHg], and five had severe LV dysfunction (LVEDP > 20 mmHg). Rats with severe LV dysfunction had significant pulmonary congestion and were likely in a chronic state of compensated congestive failure as indicated by an approximately twofold increase in both lung and right ventricle weight. Run time to fatigue and maximal oxygen uptake (V˙o 2 max) were significantly reduced (↓39 and ↓28%, respectively) in the rats with severe LV dysfunction and correlated with the magnitude of LV dysfunction as indicated by LVEDP (run time: r = 0.60, n = 21, P < 0.01 and V˙o 2 max: r = 0.93, n = 13, P < 0.01). In addition, run time to fatigue was significantly correlated withV˙o 2 max ( r = 0.87, n = 15, P < 0.01). The concentration of [3H]ouabain binding sites (Bmax) was significantly reduced (21–28%) in the three muscles comprised primarily of oxidative fibers [soleus: 259 ± 14 vs. 188 ± 17; plantaris: 295 ± 17 vs. 229 ± 18; red portion of gastrocnemius: 326 ± 17 vs. 260 ± 14 pmol/g wet tissue wt]. In addition, Bmax was significantly correlated withV˙o 2 max (soleus: r = 0.54, n = 15, P < 0.05; plantaris: r = 0.59, n = 15, P < 0.05; red portion of gastrocnemius: r = 0.65, n = 15, P < 0.01). These results suggest that downregulation of Na+-K+ pumps that possess a high affinity for ouabain in oxidative skeletal muscle may play an important role in the exercise intolerance that attends severe LV dysfunction in CHF.

Characterization of intestinal smooth muscle responses and binding sites for endothelin

1992 ◽  
Vol 70 (3) ◽  
pp. 377-384 ◽  
Author(s):  
Gordon T. Bolger ◽  
Francine Liard ◽  
Michel Garneau ◽  
Jorge Jaramillo
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Contractile Activity ◽  
Benzodiazepine Receptor ◽  
Intestinal Smooth Muscle ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Receptor Antagonists ◽  
High Affinity Binding ◽  
High Affinity

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 ± 1.3 × 10−9 M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 ± 0.8 × 10−8 M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 ± 0.6 × 10−10 M and 2138 ± 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min−1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (kl value of 0.011 min−1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10−6 M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10−6 M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10−5 M). Incubation of GPILM with ET-1 (2 × 10−8 M) for 10 min followed by washing of the tissue for 1 h resulted in a significant (p < 0.05 unpaired Student's t-test) reduction (33%) of 125I-labelled ET-1 binding that partially recovered following 2 h of washing the tissue. These results demonstrate that ET-1 is an intestinal smooth muscle spasmogen that produces its pharmacologic effects by a mechanism(s) that is not shared by other major intestinal neurotransmitters. Furthermore, intestinal smooth muscle contains specific high-affinity binding sites that likely mediate the contractile responses to ET-1.Key words: intestine, smooth muscle, endothelin, calcium channels, contraction.

scholarly journals Heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic smooth-muscle cells

1997 ◽  
Vol 326 (3) ◽  
pp. 661-668 ◽  
Author(s):  
Françoise BONO ◽  
Patrice RIGON ◽  
Isabelle LAMARCHE ◽  
Pierre SAVI ◽  
Véronique SALEL ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Muscle Cells ◽  
Platelet Factor ◽  
Protamine Sulphate ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
High Affinity

Basic fibroblast growth factor (bFGF) and its specific receptors have diverse roles on a variety of cell types, such as the induction of vascular smooth-muscle cell proliferation which contributes to restenosis after coronary balloon angioplasty. bFGF is also known to interact with heparan sulphate proteoglycans present on the cell surface or in the extracellular matrix. In this study, the binding of 125I-bFGF to human aortic smooth-muscle cells was investigated. 125I-bFGF binding to these cells was reversible and saturable. Scatchard analysis revealed the presence of two distinct binding sites: a high-affinity receptor (Kd = 38±7 pM; 1480±220 sites/cell) and a low-affinity non-saturable binding site (Kd= 8.0±2.0 nM). Pretreatment of the cells with heparinase resulted in a large reduction of 125I-bFGF binding to its low-affinity receptors, suggesting that they are heparin-like molecules. The specificity of the low- and high-affinity binding sites for bFGF was determined with acidic FGF, platelet-derived growth factor-BB and epidermal growth factor, which did not compete for 125I-bFGF binding. Expression of FGF receptor isoforms analysed by reverse transcriptase-PCR revealed the presence of only the type-1 receptor. Binding to low-affinity binding sites was antagonized by heparin, suramin, protamine sulphate and platelet factor 4. Unexpectedly, these molecules also reduced the binding of 125I-bFGF to its high-affinity sites. Consistent with these results, heparin, suramin, protamine sulphate and platelet factor 4 inhibited bFGF-induced proliferation of human aortic smooth-muscle cells. Heparin abrogated bFGF-induced release of tissue-type plasminogen activator by these cells. These observations suggest that the interaction of bFGF with human aortic smooth-muscle cells is different from that described for other cells such as endothelial cells, in which heparin acts as a potentiating factor of the mitogenic activity of bFGF.

Age-associated differential expression of Na+-K+-ATPase subunit isoforms in skeletal muscles of F-344/BN rats

1999 ◽  
Vol 87 (3) ◽  
pp. 1132-1140 ◽  
Author(s):  
Xiwu Sun ◽  
Murali Nagarajan ◽  
Philip W. Beesley ◽  
Yuk-Chow Ng
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Binding Sites ◽  
Skeletal Muscles ◽  
Brown Norway ◽  
Muscle Type ◽  
Ouabain Binding ◽  
High Affinity ◽  
Old Rats ◽  
Subunit Isoforms

Skeletal muscle expresses multiple isoforms of the Na+-K+-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na+-K+pump function. The present study tests the hypothesis that advancing age is associated with altered Na+-K+-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na+-K+-ATPase activity, measured by K+-stimulated 3- O-methylfluorescein phosphatase activity, increased by ∼50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased α1- and β1-subunit, and decreased α2- and β2-subunit in red and white Gast. In soleus, there were similar changes in expression of α1- and α2-subunits, but levels of β1-subunit were unchanged. Functional Na+-K+-ATPase units, measured by [3H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of α2-isozyme, increased in 30-mo-old rats despite decreased levels of α2-subunit. In white Gast, by contrast, decreased levels of α2-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na+-K+-ATPase activity and patterns of expression of α- and β-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.

Effect of acidosis on tension and [Ca2+]iin rat cerebral arteries: is there a role for membrane potential?

1998 ◽  
Vol 274 (2) ◽  
pp. H655-H662 ◽  
Author(s):  
Hong-Li Peng ◽  
Peter E. Jensen ◽  
Holger Nilsson ◽  
Christian Aalkjær
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Isometric Force ◽  
Cerebral Arteries ◽  
Bathing Solution ◽  
Hypercapnic Acidosis ◽  
Small Arteries ◽  
Intracellular Ca ◽  
Intracellular Microelectrodes

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+ concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i(fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing[Formula: see text] or reducing[Formula: see text] of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2′,7′-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]iis in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]idiffers between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.

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