[Na]i modulates isoproterenol's effect on Ca permeability in cultured heart cells

1987 ◽  
Vol 253 (2) ◽  
pp. C253-C262 ◽  
Author(s):  
D. Kim ◽  
T. W. Smith
Keyword(s):  
Resting Membrane Potential ◽  
Heart Cells ◽  
Ca Channel ◽  
Control Medium ◽  
Free Medium ◽  
Cardiac Muscle Cells ◽  
45Ca Uptake ◽  
Ca Uptake ◽  
Chick Heart ◽  
In Cells

Isoproterenol (ISO) augments the slow inward Ca current in cardiac muscle cells. We examined the role of intracellular Na (Nai) on ISO-mediated alterations in Ca uptake in cultured chick heart cells. In 140 mM Na medium, 1 microM ISO did not measurably alter 45Ca uptake. When cells were first preincubated in Na-free medium for 5 min and then incubated in control medium with 45Ca, ISO increased 45Ca uptake by 30%. Nifedipine (10 microM), verapamil (1 microM), or dl-propranolol (1 microM) abolished the effect of ISO on 45Ca uptake. CGP 28392 (1 microM), a Ca channel agonist, increased Ca influx in a manner that was augmented by decreased Nai, similar to the ISO response. Neither ISO nor CGP 28392 altered 45Ca uptake when cells preincubated in Na-free medium were further incubated in Na-free medium containing 45Ca. Exposure of cells to Na-free medium or 25 mM K+ medium caused depolarization of the resting membrane potential to approximately -40 mV. In the absence of ISO, the 45Ca uptake in cells preincubated in Na-free or 25 mM extracellular K (Ko) medium was significantly greater than in cells preincubated in control medium. This appeared to be due partly to increased 45Ca uptake via nifedipine-sensitive pathways. These findings support the hypothesis that reduction in Nai concentration ([Na]i) enhances the ISO-induced augmentation of Ca uptake via nifedipine-sensitive pathways (presumably via slow Ca channels), probably by a direct effect on the channels.

scholarly journals Transmembrane chloride flux in tissue-cultured chick heart cells.

1983 ◽  
Vol 81 (5) ◽  
pp. 731-748 ◽  
Author(s):  
D Piwnica-Worms ◽  
R Jacob ◽  
C R Horres ◽  
M Lieberman
Keyword(s):  
Cardiac Muscle ◽  
Rate Constant ◽  
Coulometric Titration ◽  
Chloride Concentration ◽  
Myocardial Cell ◽  
Heart Cells ◽  
Cell Water ◽  
Cardiac Muscle Cells ◽  
Diffusion Limitations ◽  
Chick Heart

To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( [Cl] i) and transmembrane 36Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, [Cl]i was 25.1 +/- 7.3 mmol x (liter cell water)-1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular 36Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min-1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent.

scholarly journals Na/H exchange in cultured chick heart cells. pHi regulation.

1985 ◽  
Vol 85 (1) ◽  
pp. 43-64 ◽  
Author(s):  
D Piwnica-Worms ◽  
R Jacob ◽  
C R Horres ◽  
M Lieberman
Keyword(s):  
Cardiac Muscle ◽  
Atomic Absorption Spectrophotometry ◽  
Heart Cells ◽  
Free Solution ◽  
Cardiac Muscle Cells ◽  
Acid Load ◽  
Chick Heart ◽  
Ph Stat ◽  
Na Uptake ◽  
Phi Regulation

The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation. The kinetics of pHi changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry; transmembrane H+ movements were evaluated under pH stat conditions. After induction of an intracellular acid load by pretreatment with NH4Cl, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na+ and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1:1. Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation. Acid-induced net Na+ uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pHo; Na+ uptake was unaffected by tetrodotoxin (10 micrograms/ml), quinidine (10(-3) M), DIDS (10(-4) M), Clo-free solution, or HCO3-free solution. Amiloride (10(-3) M) maximally inhibited both pHi regulation and Na+ uptake; the ID50 for amiloride inhibition of Na+ uptake was 3 microM. Nao-dependent H+ extrusion showed half-maximal activation at 15 mM Nao; Li+, but not K+ or choline+, could substitute for Na+ to support H+ extrusion. Cao-free solution also stimulated acid-induced Na+ uptake. We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 X s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.

Coupled sodium-calcium transport in cultured chick heart cells

1986 ◽  
Vol 250 (3) ◽  
pp. C442-C452 ◽  
Author(s):  
E. Murphy ◽  
D. M. Wheeler ◽  
A. LeFurgey ◽  
R. Jacob ◽  
L. A. Lobaugh ◽  
...  
Keyword(s):  
Fold Increase ◽  
Total Cell ◽  
Heart Cells ◽  
Electrochemical Gradient ◽  
X Ray ◽  
Sodium Calcium ◽  
Ca Uptake ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Intracellular Organelles

In cultured embryonic chick heart cells, alterations of extracellular Na (Nao) and Ca (Cao), intracellular Na (Nai) and Ca, extracellular pH, and membrane potential resulted in changes in Na and Ca contents that were consistent with sarcolemmal Na-Ca exchange. 24Na efflux measurements revealed a large ouabain-insensitive component, one-third of which was inhibited by removal of Cao. Incubating the cells in Na-free solution resulted in a rapid, 1.5- to 2-fold increase in total cell Ca that remained elevated for at least 15 min. Cells exposed for 15 min to Nao less than or equal to 20 mM became maximally loaded with Ca, whereas Ca loading fell off sharply at values of Nao greater than 20 mM. The movement of Na against its electrochemical gradient was shown to be associated with Ca accumulation. During Na-K pump inhibition (in 10(-4) M ouabain), Na initially rose 2- to 3-fold to a level below its equilibrium value; then, lowering Cao for 30 min from 1.25 to 0.75 mM caused a 26% elevation in Nai, whereas raising Cao from 1.25 to 2.7 mM resulted in a 25% fall in Nai against its electrochemical gradient. These data are consistent with Nai being maintained by a Na-Ca exchange during Na-K pump inhibition. In the presence of ouabain (10(-4) M), Ca uptake into intracellular organelles, e.g., mitochondria, was suggested by an increase in total cell Ca as well as the occurrence of mitochondrial matrix granules, which were shown qualitatively by X-ray analysis to contain Ca. Although matrix granules also occurred in mitochondria during Na-free incubation, they did not contain detectable amounts of Ca when examined under identical conditions of fixation and analysis.

Altered Ca fluxes and contractile state during pH changes in cultured heart cells

1987 ◽  
Vol 253 (1) ◽  
pp. C137-C146 ◽  
Author(s):  
D. Kim ◽  
T. W. Smith
Keyword(s):  
Negative Inotropic Effect ◽  
Inotropic Effect ◽  
Heart Cells ◽  
45Ca Uptake ◽  
Contractile State ◽  
Ventricular Cells ◽  
Ca Uptake ◽  
Cell Motion ◽  
Induced Changes ◽  
Cultured Heart Cells

We studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pHi) or extracellular (pHo) changes in pH using cultured chick embryo ventricular cells. A change in pHo of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45Ca uptake at 10 s by -29 or +22%, respectively. The pHo-induced change in Ca uptake was not sensitive to nifedipine (10 microM), but was Na gradient dependent. Changes in pHi produced by NH4Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45Ca uptake or efflux. However, larger changes in pHi were associated with altered Ca uptake. Changes in pHo from 7.4 to 6.0 or to 8.8 were associated with initial changes in 45Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH4Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH4Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationship between pHi and contractile state. These results indicate that pHo-induced changes in contractile state in cultured heart cells are closely correlated with altered transsarcolemmal Ca movements and presumably are due (at least in part) to these Ca flux changes. In contrast, pHi-induced changes in contractile state appear principally to involve altered Ca handling within the cell and/or altered Ca sensitivity of myofibrils.

Techniques for cryoultramicrotomy of propane jet frozen biological samples

1986 ◽  
Vol 44 ◽  
pp. 260-261
Author(s):  
B. Craig ◽  
L. Hawkey ◽  
A. LeFurgey
Keyword(s):  
Freeze Fracture ◽  
Freeze Substitution ◽  
Heart Cells ◽  
Crystal Formation ◽  
X Ray ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
A New Technique

Ultra-rapid freezing followed by cryoultramicrotomy is essential for the preservation of diffusible elements in situ within cells prior to scanning transmission electron microscopy and quantitative energy dispersive x-ray microanalysis. For cells or tissue fragments in suspension and for monolayer cell cultures, propane jet freezing provides cooling rates greater than 30,000°C/sec with regions up to 40μm in thickness free of significant ice crystal formation. While this method of freezing has frequently been applied prior to freeze fracture or freeze substitution, it has not been widely utilized prior to cryoultramicrotomy and subsequent x-ray microanalytical studies. This report describes methods devised in our laboratory for cryosectioning of propane jet frozen kidney proximal tubule suspensions and cultured embryonic chick heart cells, in particular a new technique for mounting frozen suspension specimens for sectioning. The techniques utilize the same specimen supports and sample holders as those used for freeze fracture and freeze substitution and should be generally applicable to any cell suspension or culture preparation.

scholarly journals Trypsin-induced increase in cyclic AMP concentration in rat thymocytes An effect independent of calcium and calmodulin

1986 ◽  
Vol 239 (3) ◽  
pp. 603-607 ◽  
Author(s):  
J Segal
Keyword(s):  
Cyclic Amp ◽  
Free Medium ◽  
Standard Medium ◽  
Intracellular Pool ◽  
Calmodulin Inhibitor ◽  
Related Increase ◽  
In Cells

Trypsin produces a dose-related increase in cellular cyclic AMP concentration in rat thymocytes [Shneyour, Patt & Trainin (1976) J. Immunol. 117, 2143-2149; Segal & Ingbar (1983) Clin. Res. 31, 277A]. In the present study, I examined whether this effect of trypsin requires Ca2+ and whether it is modified by calmodulin. In fresh thymocytes suspended in standard medium (containing 1 mM-Ca2+), trypsin produced a concentration-dependent increase in cytoplasmic free Ca2+ concentration, which was evident at a concentration of 50 micrograms of trypsin/ml and reached maximal values at about 1 mg/ml. This effect of trypsin was very prompt in onset, almost immediate, and reached maximal values within 2-3 min. But in cells suspended in essentially Ca2+-free medium (6 nM free Ca2+), trypsin had no effect on cytoplasmic free Ca2+ concentration, which indicates that trypsin acted by increasing Ca2+ uptake rather than Ca2+ release from an intracellular pool. However, the increase in thymocyte cyclic AMP concentration produced by trypsin was independent of extracellular Ca2+ and was not influenced by calmodulin, because it was the same in the presence or absence of Ca2+ and was not changed by the calmodulin inhibitor trifluoperazine. I therefore suggest that in rat thymocytes the trypsin-induced increase in cyclic AMP concentration does not require Ca2+ and is not influenced by calmodulin.

FURTHER STUDIES ON THE DUAL REQUIREMENT FOR PHENYLALANINE AND TYROSINE IN TISSUE CULTURE

1961 ◽  
Vol 39 (5) ◽  
pp. 925-932 ◽  
Author(s):  
Helen J. Morton ◽  
Joseph F. Morgan
Keyword(s):  
Amino Acids ◽  
Tissue Culture ◽  
Chick Embryo ◽  
Heart Cells ◽  
Present System ◽  
Chick Heart ◽  
High Concentrations ◽  
Chick Embryo Heart ◽  
Embryo Heart ◽  
Related Compounds

Seventeen structurally related compounds were tested for their ability to substitute for phenylalanine or tyrosine in the nutrition of chick embryo heart fragments. DL-Alanyl-DL-phenylalanine replaced phenylalanine. All other compounds had negligible effects, and most were toxic at high concentrations. β-Phenylserine, a phenylalanine antagonist, actually prolonged the survival of chick heart cells but only if both phenylalanine and tyrosine were present. Similarly, optimal reversal of β-phenylserine toxicity was dependent on the presence of both amino acids. Although phenylalanine and tyrosine are not interconvertible in the present system, it has been shown that three phenylalanine antagonists, p-fluorophenylalanine, β-2-thienylalanine, and β-phenylserine, can be identified by their relationship to tyrosine, rather than to phenylalanine.

scholarly journals Agonist-induced Ca2+ influx in human neutrophils is secondary to the emptying of intracellular calcium stores

1991 ◽  
Vol 277 (1) ◽  
pp. 73-79 ◽  
Author(s):  
M Montero ◽  
J Alvarez ◽  
J Garcia-Sancho
Keyword(s):  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Time Course ◽  
Human Neutrophils ◽  
Calcium Stores ◽  
Free Medium ◽  
Prolonged Incubation ◽  
Intracellular Calcium Stores ◽  
Cytochrome P 450 ◽  
In Cells

Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca(2+)-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B4 released different amounts of calcium from the stores and induced Ca2+ (Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca(2+)-free medium, but returned to basal levels in cells incubated in Ca(2+)-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2+ (Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].

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