Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells

1989 ◽  
Vol 256 (4) ◽  
pp. C786-C792 ◽  
Author(s):  
J. C. Allen ◽  
S. S. Navran ◽  
C. L. Seidel ◽  
D. K. Dennison ◽  
J. M. Amann ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Muscle Cells ◽  
Ouabain Binding ◽  
Na Pump ◽  
Site Number

Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [3H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.

MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Jana Holmar ◽  
Heidi Noels ◽  
Joachim Jankowski ◽  
Setareh Orth-Alampour
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Incubation Time ◽  
Fetal Calf Serum ◽  
Ex Vivo ◽  
Calf Serum

Abstract Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

Factor Xa Generation at the Surface of Cultured Rat Vascular Smooth Muscle Cells in an in Vitro Flow System

1998 ◽  
Vol 120 (4) ◽  
pp. 484-490 ◽  
Author(s):  
C. L. Hall ◽  
M. B. Taubman ◽  
Y. Nemerson ◽  
V. T. Turitto
Keyword(s):  
Smooth Muscle ◽  
Shear Rate ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Transport Theory ◽  
Calf Serum ◽  
Factor Xa ◽  
Muscle Cells ◽  
Wall Shear

The purpose of the present investigation was to explore the effects of well-defined flow conditions on the activity of tissue factor (TF) expressed on the surface of cultured rat vascular smooth muscle cells. Cells were cultured to confluence on Permanox brand slides and stimulated to express TF by a 90 min incubation with fresh growth medium containing 10 percent calf serum. The stimulated cells were then placed in a parallel plate flow chamber and perfused with Hank’s Balanced Salt Solution containing factor VIIa, factor X (FX), and calcium. The chamber effluent was collected and assayed for factor Xa (FXa) and the steady-state flux of FXa was calculated. The flux values were 68.73, 94.81, 139.75, 138.19, 316.82, and 592.92 fmole/min/cm2 at wall shear rates of 10, 20, 40, 80, 320, and 1280 s−1 respectively. The FXa flux depended on the wall shear rate to a greater degree than predicted by classical mass transport theory. The flux at each shear rate was three to five times less than that calculated according to the Leveque solution. These features of the experimental data imply nonclassical behavior, which may partially result from a direct effect of flow on the cell layer.

Intracellular free Na+ in resting and activated A7r5 vascular smooth muscle cells

1993 ◽  
Vol 264 (6) ◽  
pp. C1513-C1524 ◽  
Author(s):  
M. L. Borin ◽  
W. F. Goldman ◽  
M. P. Blaustein
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cell Activation ◽  
Prolonged Exposure ◽  
Muscle Cells ◽  
Resting Cells ◽  
Free Medium ◽  
Na Pump

Regulation of intracellular Na+ ([Na+]i) in cultured vascular smooth muscle cells (A7r5 line) was studied with Na(+)-sensitive fluorescent dye sodium-binding benzofuran isophthalate. Digital imaging microscopy was used to study single-cell fluorescence. Na+ was distributed uniformly in cytoplasm and nucleus; mean Na+ concentration in resting cells was 4.4 +/- 0.3 mM in cytoplasmic areas ([Na+]cyt) and 4.5 +/- 0.4 mM in nuclear areas ([Na+]n). Na+ pump inhibition and cell activation evoked uniform changes in [Na+]cyt and [Na+]n. Inhibition of Na+ pump with 1 mM ouabain or K(+)-free medium caused a rise in [Na+]cyt; in the latter case, [Na+]cyt fell rapidly when external K+ was later restored. Exposure to Ca(2+)-free medium also caused [Na+]cyt to rise; this effect was augmented by Na+ pump inhibition and was reversed by 10(-5) M verapamil or nitrendipine or by restoration of external Ca2+. The implication is that this Na+ entry in absence of external Ca2+ is mediated by Ca2+ channels. Activation by 10(-9) M arginine vasopressin (AVP) and 10(-6) M serotonin (5-HT) caused [Na+]cyt to increase, but response to 5-HT was small (0.6 mM on average) and transient, whereas response to AVP was larger (2.4 mM on average) and was maintained as long as AVP was present (to 20 min). AVP and, to a much smaller extent, 5-HT stimulated Na+ influx; this could be detected when Na+ pump was inhibited by ouabain. Both AVP and 5-HT activated the Na+ pump, as detected by ouabain-sensitive decrease in [Na+]cyt when Na+ influx was inhibited. Agonist-evoked increases in [Na+]cyt were dependent on a rise in cytosolic Ca2+ concentration ([Ca2+]cyt); these [Na+]cyt responses were abolished by prolonged exposure to Ca(2+)-free media, when cytoplasmic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or when Ca2+ mobilization was blocked with thapsigargin. Raising [Ca2+]cyt with 40 mM K+ or with thapsigargin did not increases in [Na+]cyt. We conclude that 1) AVP- and 5-HT-evoked increases in [Na+]cyt are agonist specific and depend on the balance between stimulated Na+ influx and efflux; 2) AVP and 5-HT activate the Na+ pump; this is, at least in part, independent of agonist-induced rise in [Na+]cyt; and 3) a rise in [Ca2+]cyt is necessary but not sufficient to trigger agonist-evoked rise in [Na+]i.

Cultured rat vascular smooth muscle cells: extracellular calcium and Na+-K+ regulation

1985 ◽  
Vol 248 (5) ◽  
pp. C436-C441 ◽  
Author(s):  
M. Kino ◽  
A. Tokushige ◽  
H. Tamura ◽  
L. Hopp ◽  
B. M. Searle ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Extracellular Calcium ◽  
Intracellular Sodium ◽  
Intracellular Potassium ◽  
Na Pump ◽  
Deficient Medium

This study explores the relationship between extracellular calcium (Cao) and Na+-K+ regulation as it particularly pertains to the activity of the Na+ pump in cultured vascular smooth muscle cells (VSMCs) originating from Sprague-Dawley rats. As compared with cells incubated in media containing 0.5, 2.0, or 4.0 mM calcium, when the Na pump is active, VSMCs incubated in a Ca-deficient medium show a marked increase in intracellular sodium and no significant change in intracellular potassium. Associated with the rise in intracellular sodium there is an augmented activity of the Na pump. When the Na pump is inhibited, VSMCs incubated in either high-Ca medium (Cao = 4.0 mM) or Ca-deficient medium manifest a greater decline in intracellular potassium than cells incubated in media containing 0.5 or 2.0 mM calcium. Furthermore, when the Na pump is inhibited, VSMCs incubated in a Ca-deficient medium exhibit higher intracellular sodium levels in comparison with their counterparts incubated in media containing calcium. Flux experiments indicate that the aforementioned changes reflect increased membrane permeabilities to Na+ and K+. It is concluded that by regulating the permeability of the VSMC membrane, Cao plays an important role in the intracellular Na+-K+ homeostasis and that its effect on the Na pump is mediated via perturbations in the intracellular Na+ and K+ concentrations.

[3H]ouabain binding to cultured rat vascular smooth muscle cells

1984 ◽  
Vol 246 (5) ◽  
pp. C551-C557 ◽  
Author(s):  
F. Khalil ◽  
L. Hopp ◽  
B. M. Searle ◽  
A. Tokushige ◽  
H. Tamura ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dissociation Constant ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Hill Coefficient ◽  
Equilibrium Dissociation Constant ◽  
Na Pump ◽  
Equilibrium Dissociation

The number of Na+ pump units (Bmax) and the equilibrium dissociation constant (Kd) for ouabain as well as parameters of K+ binding to the Na+ pump were examined in in vitro-grown vascular smooth muscle cells ( VSMC ) derived from Sprague-Dawley rats. The technique to measure these variables utilizes analyses of [3H]ouabain displacement from its VSMC receptors by nonlabeled ouabain and K+. The mean values for Bmax and Kd in the cultured VSMCs were 1.95 X 10(5) receptor sites per single VSMC and 2.68 X 10(-6) M, respectively. The equilibrium dissociation constant for K+ (Ki) was 0.92 mM. K+ binding to the cultured VSMCs demonstrated positive cooperativity with a Hill coefficient (n) of 1.78.

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