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.

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 Co-culture of embryonic chick heart cells and ciliary ganglia induces parasympathetic responsiveness in embryonic chick heart cells

1993 ◽  
Vol 292 (2) ◽  
pp. 395-399 ◽  
Author(s):  
J V Barnett ◽  
M Taniuchi ◽  
M B Yang ◽  
J B Galper
Keyword(s):  
G Proteins ◽  
Fold Increase ◽  
Heart Cell ◽  
Heart Cells ◽  
Embryonic Chick ◽  
In Ovo ◽  
Chronotropic Response ◽  
Muscarinic Stimulation ◽  
Chick Heart ◽  
Embryonic Chick Heart

We have developed a system for the co-culture of embryonic chick heart cells obtained from embryos at 3.5 days in ovo with ciliary ganglia from chick embryos at 7 days in vivo. After 3 days of co-culture, removal of the ciliary ganglia resulted in complete degeneration of axons within 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsive to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropic response to muscarinic stimulation (paired t test, P < 0.02) which persisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increase in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha 39 (39 kDa subunit) and a 2.5-fold increase in the level of alpha 41. The level of the G-protein subunit alpha s remained unchanged. Culture of embryonic chick heart cells at 3.5 days in ovo with medium conditioned by the growth of embryonic chick heart cells and ciliary ganglia had an effect on the chronotropic response to muscarinic stimulation and on alpha 39 and alpha 41 levels identical to that of co-culture. These data suggest that a soluble factor released during the co-culture of embryonic chick heart cells and ciliary ganglia is capable of inducing muscarinic responsiveness. These studies suggest that innervation of the heart may induce parasympathetic responsiveness by increasing the availability of G-proteins which couple the muscarinic receptor to a physiological response.

Calcium elevation in cultured heart cells: its role in cell injury

1983 ◽  
Vol 245 (5) ◽  
pp. C316-C321 ◽  
Author(s):  
E. Murphy ◽  
J. F. Aiton ◽  
C. R. Horres ◽  
M. Lieberman
Keyword(s):  
Cell Injury ◽  
Fold Increase ◽  
Myocardial Cell ◽  
Heart Cells ◽  
Atp Content ◽  
Intracellular Enzyme ◽  
Fourfold Increase ◽  
Myocardial Cell Injury ◽  
Chick Heart ◽  
Embryonic Chick Heart

Inhibition of the Na+-K+ pump in cultured embryonic chick heart cells promotes the elevation of intracellular Ca2+ and is a useful manipulation to study the relationship between Ca2+ and myocardial cell injury. One hour of Na+-K+ pump inhibition resulted in a fourfold increase in cell Na+, a 50% decline in cell K+, and a 5- to 10-fold increase in cell Ca2+, 45% of which is mitochondrial. The degree of cell injury induced by Ca2+ loading was evaluated by monitoring the content of adenosine 5'-triphosphate (ATP) and the release of the intracellular enzyme lactate dehydrogenase (LDH). Under these conditions ATP content declined by 25-30% and LDH release increased from 1 to 1.4% of the total LDH. Furthermore, cells subjected to 1 h of Na+-K+ pump inhibition and returned to control solution for 5 h showed that Ca2+ decreased to near control levels and ATP content was restored. Although inhibition of Na+-K+ transport caused a large increase in cell Ca2+, neither Na+-K+ pump inhibition nor elevation in total cell Ca2+ per se resulted in irreversible myocardial cell injury.

(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells

1987 ◽  
Vol 253 (5) ◽  
pp. C721-C730 ◽  
Author(s):  
S. Liu ◽  
R. Jacob ◽  
D. Piwnica-Worms ◽  
M. Lieberman
Keyword(s):  
Disulfonic Acid ◽  
Heart Cells ◽  
Free Solution ◽  
Embryonic Chick ◽  
Electrochemical Gradient ◽  
Coupled Process ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Cultured Heart Cells ◽  
Na Uptake

The coupled movements of Na, K, and Cl were studied in cultured chick embryonic heart cells using ion-selective microelectrodes. Movements of K and Cl in response to changes in extracellular [K] ([K]o) showed a furosemide-sensitive coupled process. The movement of Na was then studied. Lowering extracellular [Na] ([Na]o) to 27 mM caused a decrease in intracellular Cl activity (aicl). Upon restoring [Na]o to 143 mM, Cl was taken up against its electrochemical gradient (delta mu Cl). In Cl-free solution, cells lost Na against delta mu Na and simultaneously lost Cl. Upon restoring extracellular [Cl] ([Cl]o), Cl was taken up against delta mu Cl; this was accompanied by an uptake of Na. The Cl uptake was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-insensitive (0.1 mM) but inhibited by removing Nao. Both Cl and Na uptakes were potentiated by raising [K]o from 5.4 to 15 mM, and Na uptake was diminished by lowering [K]o to 1 mM. In all experiments, Cl and Na movements were furosemide (0.3 mM) or bumetanide-sensitive (0.1 mM). Removal of Nao, with resultant depletion of intracellular [Na] ([Na]i), blocked the furosemide or bumetanide-sensitive Cl loss or uptake upon exposure to zero or 133 mM [K]o + SITS (0.1 mM), respectively. These results suggest that cultured heart cells possess an electroneutral (Na + K + 2Cl) cotransport.

Cytosolic free magnesium concentration in cultured chick heart cells

1989 ◽  
Vol 257 (1) ◽  
pp. C141-C146 ◽  
Author(s):  
S. Rotevatn ◽  
E. Murphy ◽  
L. A. Levy ◽  
B. Raju ◽  
M. Lieberman ◽  
...  
Keyword(s):  
Salt Solution ◽  
Significant Proportion ◽  
Fold Increase ◽  
Magnesium Concentration ◽  
Heart Cells ◽  
Ion Gradient ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Free Magnesium ◽  
Solution Changes

Cytosolic free magnesium (Mgi) was measured in embryonic chick heart cells loaded with one of two newly developed 19F nuclear magnetic reasonance (NMR)-sensitive magnesium chelators, 4-methyl,5-fluoro-2-aminophenol-N,N,O-triacetate (MF-APTRA) and 5-fluoro-2-aminophenol-N,N,O-triacetate (5F-APTRA). The cells, embedded in strands of collagen, were superfused at a rate that allowed for solution changes in 2 min. In this preparation 19F- and 31P-NMR spectra were stable for at least 3.5 h. Because Na-coupled Mg countertransport may be a possible mechanism of Mg transport, in some experiments extracellular Na was reduced to 1 mM (choline substituted). This manipulation caused a 2.5-fold increase in Mgi from the basal level of 0.56 mM. A significant proportion of this increase in Mgi could be secondary to an increase in Cai that occurs with low extracellular Na (Nao) perfusion (Nai-Cao exchange). Perfusing cells with nominally Ca-free, 1 mM Na salt solution substantially attenuated the increase in Mgi that occurred with Ca present (1.25 mM) in the low Na (1 mM) solution. Furthermore, perfusion with 1 mM Na, Mg-free salt solution caused a 1.5-fold increase in Mgi, which cannot be attributable to Nai-Mgo exchange. Therefore attempts to describe the regulation of Mgi in heart cells must differentiate between the effects of Nai-Mgo exchange and competition for binding sites that are secondary to stimulation of ion gradient-coupled mechanisms.

[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.

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