Glycolytic flux in permeabilized freshly isolated vascular smooth muscle cells

1998 ◽  
Vol 274 (1) ◽  
pp. C88-C96 ◽  
Author(s):  
Christopher D. Hardin ◽  
Dorian R. Finder
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Carotid Arteries ◽  
Dextran Sulfate ◽  
Lactate Production ◽  
Glycolytic Flux ◽  
Bathing Solution ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Glycolytic Intermediates

To determine whether channeling of glycolytic intermediates can occur in vascular smooth muscle (VSM), we permeabilized freshly isolated VSM cells from hog carotid arteries with dextran sulfate. The dextran sulfate-treated cells did not exclude trypan blue, a dye with molecular weight of ∼1,000. If glycolytic intermediates freely diffuse, plasmalemmal permeabilization would allow intermediates to exit the cell and glycolytic flux should cease. We incubated permeabilized and nonpermeabilized cells with 5 mM [1-13C]glucose at 37°C for 3 h. 13C nuclear magnetic resonance (NMR) was used to determine relative [3-13C]lactate production and to identify any13C-labeled glycolytic intermediates that exited from the permeabilized cells. [3-13C]lactate production from [1-13C]glucose was decreased by an average of 32% ( n = 6) in permeabilized cells compared with intact cells. No13C-labeled glycolytic intermediates were observed in the bathing solution of permeabilized cells. We conclude that channeling of glycolytic intermediates can occur in VSM cells.

scholarly journals A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle

2010 ◽  
Vol 299 (5) ◽  
pp. C988-C993 ◽  
Author(s):  
Hak Rim Kim ◽  
Paul C. Leavis ◽  
Philip Graceffa ◽  
Cynthia Gallant ◽  
Kathleen G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Direct Detection ◽  
New Method ◽  
Actin Dynamics ◽  
Tat Peptide ◽  
Isolated Cells ◽  
Intact Cells ◽  
Permeabilized Cells

Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca2+ concentration transients and signal transduction on actin dynamics in intact cells.

scholarly journals NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

2000 ◽  
Vol 279 (6) ◽  
pp. H2872-H2878 ◽  
Author(s):  
John T. Barron ◽  
Liping Gu ◽  
Joseph E. Parrillo
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Redox Potential ◽  
Redox State ◽  
Lactate Production ◽  
Glycolytic Flux ◽  
Dihydroxyacetone Phosphate ◽  
Redox Potentials ◽  
Redox Couples ◽  
Lactate And Pyruvate

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na+-K+-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na+-K+-ATPase by 10 μM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H+ transporter secondary to decreased availability of H+ for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H+ transporter, which was specifically linked to reduced activity of Na+-K+-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na+-K+-ATPase.

Fatty acid, tricarboxylic acid cycle metabolites, and energy metabolism in vascular smooth muscle

1994 ◽  
Vol 267 (2) ◽  
pp. H764-H769 ◽  
Author(s):  
J. T. Barron ◽  
S. J. Kopp ◽  
J. Tow ◽  
J. E. Parrillo
Keyword(s):  
Smooth Muscle ◽  
Fatty Acid ◽  
Energy Metabolism ◽  
Carotid Artery ◽  
Vascular Smooth Muscle ◽  
Lactate Production ◽  
Tca Cycle ◽  
High Energy ◽  
Tricarboxylic Acid ◽  
O2 Consumption

The influence of octanoate on O2 consumption, tricarboxylic acid (TCA) cycle intermediates, and high-energy phosphates was examined in intact resting porcine carotid artery to investigate the role of fatty acid in energy metabolism and its integration with glucose metabolism in vascular smooth muscle. Incubation of resting arteries with octanoate (0.5 mM), which was previously shown to inhibit aerobic glycolysis (6), inhibited lactate production by 64% and increased O2 consumption by 30%. The increase in O2 consumption with octanoate was approximately equal to that calculated to account for the ATP production lost by inhibition of aerobic lactate production by octanoate. In glucose-free medium, the level of high-energy phosphate was reduced but was restored when octanoate was included in the incubation medium. This was associated with an increase in O2 consumption. These results suggest that the energy requirements of resting carotid artery can be largely met by the oxidative metabolism of fatty acid. Octanoate induced anaplerosis of the TCA cycle, as indicated by a 70% increase in the level of citrate. Extracellular glucose was necessary for octanoate-induced anaplerosis, probably by providing the extra carbon via pyruvate carboxylation, whereas a coupled transamination involving aspartate was a less important anaplerotic mechanism.

Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells

2000 ◽  
Vol 278 (4) ◽  
pp. C803-C811 ◽  
Author(s):  
Pamela G. Lloyd ◽  
Christopher D. Hardin
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Major Product ◽  
Intact Cells ◽  
Glycolytic Intermediate ◽  
Cerebral Microvessels ◽  
Permeabilized Cells ◽  
Direct Contrast ◽  
Cerebrovascular Smooth Muscle Cells ◽  
Fructose 1,6 Bisphosphate

We used β-escin-permeabilized pig cerebral microvessels (PCMV) to study the organization of carbohydrate metabolism in the cytoplasm of vascular smooth muscle (VSM) cells. We have previously demonstrated (Lloyd PG and Hardin CD. Am J Physiol Cell Physiol 277: C1250–C1262, 1999) that intact PCMV metabolize the glycolytic intermediate [1-13C]fructose 1,6-bisphosphate (FBP) to [1-13C]glucose with negligible production of [3-13C]lactate, while simultaneously metabolizing [2-13C]glucose to [2-13C]lactate. Thus gluconeogenic and glycolytic intermediates do not mix freely in intact VSM cells (compartmentation). Permeabilized PCMV retained the ability to metabolize [2-13C]glucose to [2-13C]lactate and to metabolize [1-13C]FBP to [1-13C]glucose. The continued existence of glycolytic and gluconeogenic activity in permeabilized cells suggests that the intermediates of these pathways are channeled (directly transferred) between enzymes. Both glycolytic and gluconeogenic flux in permeabilized PCMV were sensitive to the presence of exogenous ATP and NAD. It was most interesting that a major product of [1-13C]FBP metabolism in permeabilized PCMV was [3-13C]lactate, in direct contrast to our previous findings in intact PCMV. Thus disruption of the plasma membrane altered the distribution of substrates between the glycolytic and gluconeogenic pathways. These data suggest that organization of the plasma membrane into distinct microdomains plays an important role in sorting intermediates between the glycolytic and gluconeogenic pathways in intact cells.

Metabolie Effects of Direct Current Stimulation on Cultured Vascular Smooth Muscle Cells

1984 ◽  
Vol 39 (11-12) ◽  
pp. 1141-1144 ◽  
Author(s):  
Helmut Heinle ◽  
Gerhard Sigg ◽  
Amo Reich ◽  
Klaus-Ulrich Thiedemann
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cell Activity ◽  
Lactate Production ◽  
Muscle Cells ◽  
Enhancing Effect ◽  
Mediating Role

Abstract Vascular smooth muscle cells from rabbit arteries were grown in tissue culture and stimulated by DC impulses (1 mA, 1V, 10 Hz, 1 ms/imp). Scanning microscopic examination disclosed that in stimulated cultures the cell surface was enlarged by numerous microvilli. This was interpreted as being indicative of an increase in cell activity. Cellular metabolism was character­ized by analyzing the incubation medium for glucose, glutamate/glutamine, and lactate. When compared to unstimulated controls, stimulation caused an increase in the uptake of glucose and glutamine as well as an increased lactate production. The enhancing effect on metabolism was prevented when the “calcium antagonist” verapamil was present (5 × 10-6ᴍ). Although the exact mechanism by which DC stimulation influences the cells remains obscure, this finding indicates an important mediating role of Ca2+ ions.

Divergent kinase signaling mediates agonist-induced phosphorylation of phosphatase inhibitory proteins PHI-1 and CPI-17 in vascular smooth muscle cells

2006 ◽  
Vol 290 (3) ◽  
pp. C892-C899 ◽  
Author(s):  
Huan Pang ◽  
Zhenheng Guo ◽  
Zhongwen Xie ◽  
Wen Su ◽  
Ming C. Gong
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Muscle Cells ◽  
Resting Cells ◽  
Kinase Signaling ◽  
Intact Cells ◽  
Agonist Stimulation

Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation.

Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

1992 ◽  
Vol 262 (5) ◽  
pp. C1258-C1265 ◽  
Author(s):  
Y. T. Xuan ◽  
O. L. Wang ◽  
A. R. Whorton
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.

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