A discrete Na-Ca exchange-dependent Ca compartment in rat ventricular cells: exchange and localization

1992 ◽  
Vol 262 (5) ◽  
pp. C1149-C1153 ◽  
Author(s):  
G. A. Langer ◽  
T. L. Rich
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Binding Sites ◽  
Rat Heart ◽  
Half Time ◽  
Adult Rat ◽  
Ventricular Cells ◽  
Intracellular Ca ◽  
Rapid Perfusion ◽  
Limited Exchange

Application of a new rapid perfusion (up to 4.8 ml/s) technique to 45Ca-labeled ventricular cells from adult rat heart has defined a discrete intracellular calcium (Ca) compartment with the following characteristics: 1) its exchange is absolutely dependent on operation of the Na-Ca exchanger, i.e., its isotopic content remains constant during washout in the absence of Na and Ca and is released only upon addition of Na and Ca to the perfusate. 2) At an extracellular Ca concentration of 1.0 mM it contains 350 mumol/kg dry wt cells and exchanges with half time of 650 ms. Ca flux from the compartment is 385 mumol.kg dry wt-1.s-1 or 20% of the total nonperfusion limited flux from the cells. 3) Its content is decreased 19% by 10 mM caffeine but not diminished by exposure of the cells to 10(-6) M ryanodine and not accessible to lanthanum (La) displacement. 4) Only limited exchange occurs when only Na or Ca is present and exchange is virtually eliminated by substitution of extracellular Li for extracellular Na. 5) Replacement of Na and Ca to the perfusate after various periods of removal produces no contraction (despite immediate Ca release from the cell). The results define a discrete intracellular Ca compartment which exchanges only via the Na-Ca exchanger. It is not La accessible, not in the ryanodine- or caffeine-sensitive portions of the sarcoplasmic reticulum, not in the mitochondria nor at the myofilaments, but may reside at inner sarcolemmal leaflet binding sites.

Effects of Na+/Ca2+ exchanger downregulation on contractility and [Ca2+]i transients in adult rat myocytes

2002 ◽  
Vol 283 (4) ◽  
pp. H1616-H1626 ◽  
Author(s):  
George M. Tadros ◽  
Xue-Qian Zhang ◽  
Jianliang Song ◽  
Lois L. Carl ◽  
Lawrence I. Rothblum ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Current Density ◽  
Resting Membrane Potential ◽  
Action Potential Amplitude ◽  
Half Time ◽  
Adult Rat ◽  
Postmyocardial Infarction ◽  
Intracellular Ca ◽  
Rat Myocytes

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na+/Ca2+exchange (NCX1) activity, altered contractility, and intracellular Ca2+ concentration ([Ca2+]i) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca2+-ATPase abundance, SR Ca2+uptake, resting membrane potential, action potential amplitude and duration, L-type Ca2+ current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca2+ concentration ([Ca2+]o) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca2+]i transient amplitudes and SR Ca2+ contents than control myocytes. At 0.6 mM [Ca2+]o, contraction and [Ca2+]i transient amplitudes and SR Ca2+ contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca2+]o, contraction and [Ca2+]i transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca2+]i transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca2+ influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.

scholarly journals Intracellular Calcium Binding and Release in Frog Heart

1973 ◽  
Vol 62 (6) ◽  
pp. 693-706 ◽  
Author(s):  
Saul Winegrad
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Binding Sites ◽  
Calcium Binding ◽  
Calcium Content ◽  
Frog Heart ◽  
Affinity Constant ◽  
Total Calcium ◽  
Abrupt Changes ◽  
Calcium Binding Sites

The capacities and affinities of intracellular calcium-binding sites have been studied in frog ventricles, in which the concentration of Ca++ in the sarcoplasm can be controlled as a result of treatment with EDTA. The total calcium content of calcium-depleted and nondepleted muscles at rest and muscles generating considerable tension was 0.8, 1.4, and 5.4 µmol/g of muscle, respectively. Net movement of calcium into or out of the cells occurred without change in tension when the sarcoplasmic concentration of Ca++ was either of two values, less than 10-7 M or approximately 5 x 10-7 M. These data can be explained by the presence of two groups of intracellular calcium sinks which compete with the contractile proteins, one with a capacity of about 0.6 µmol/g and an affinity constant greater than 107 M-1 and a second with a capacity of 4.0 µmol/g and an affinity constant of about 2 x 106 M-1. The higher affinity calcium is released by anoxia, oligomycin, or abrupt changes in sarcoplasmic Ca++. Muscles soaked in Sr-Ringer's contain electron densities in the sarcoplasmic reticulum and to a lesser extent in the mitochondria.

Subcellular localization of the delayed rectifier K+ channels KCNQ1 and ERG1 in the rat heart

2004 ◽  
Vol 286 (4) ◽  
pp. H1300-H1309 ◽  
Author(s):  
Hanne Borger Rasmussen ◽  
Morten Møller ◽  
Hans-Günther Knaus ◽  
Bo Skaaning Jensen ◽  
Søren-Peter Olesen ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Rat Heart ◽  
Ventricular Myocytes ◽  
Cardiac Cells ◽  
Delayed Rectifier ◽  
Transverse Tubular System ◽  
Adult Rat ◽  
Confocal Immunofluorescence Microscopy ◽  
Ventricular Cells ◽  
Tubular System

In the heart, several K+ channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K+ currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K+ channels, KCNQ1 and ether- a- go- go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter. Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these K+ channels to the transverse tubular system close to the Ca2+ channels renders them with maximal repolarizing effect.

Thapsigargin inhibits Ca2+ uptake, and Ca2+ depletes sarcoplasmic reticulum in intact cardiac myocytes

1993 ◽  
Vol 265 (2) ◽  
pp. H517-H522 ◽  
Author(s):  
A. M. Janczewski ◽  
E. G. Lakatta
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Rat Myocardium ◽  
Half Time ◽  
Adult Rat ◽  
Time To Peak ◽  
Rapid Pacing ◽  
Ethanesulfonic Acid

We examined the effects of thapsigargin on Ca2+ accumulation by the sarcoplasmic reticulum (SR) and on electrically stimulated beats in single adult rat ventricular myocytes loaded with indo 1 and bathed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer containing 1 mM Ca2+ at 23 degrees C. The SR Ca2+ content was assessed from the magnitude of intracellular Ca2+ (Ca2+i) transients and contractions elicited by rapid, brief applications of caffeine. After 20-30 min of exposure to 200 nM thapsigargin, the caffeine-dependent Ca2+i transients were abolished or markedly diminished (by 89 +/- 4%). The postrest potentiation of the Ca2+i transient and contraction, typical for rat myocardium, was abolished. Thapsigargin did not significantly change resting Ca2+i but diminished the amplitude of the steady-state Ca2+i transients by 73%, prolonged the time to peak by 24%, and prolonged the half-time (t1/2) of the Ca2+i transient decline by 42%. Progressive SR Ca2+ depletion by thapsigargin was strongly related (r = -0.78) to the prolongation of the t1/2 of relaxation of the steady-state Ca2+i transients, suggesting that the thapsigargin-dependent SR Ca2+ depletion results from an inhibition of the SR Ca2+ uptake. This interpretation was corroborated by comparison of the effects of thapsigargin with those of ryanodine (100 nM), which depletes SR of Ca2+ by accelerating the SR Ca2+ efflux but does not inhibit the SR Ca2+ pump. During rapid pacing (5 Hz), which raises Ca2+i and thus Ca2+ available for SR uptake, the caffeine-dependent SR Ca2+ release was restored in ryanodine-treated cells but not in the presence of thapsigargin.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

2006 ◽  
Vol 291 (6) ◽  
pp. H2790-H2800 ◽  
Author(s):  
Harjot K. Saini ◽  
Naranjan S. Dhalla
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Extracellular Ph ◽  
The Other ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cardiomyocytes ◽  
Intracellular Ca ◽  
Isolated Rat

Although the Na+/H+ exchanger (NHE) is considered to be involved in regulation of intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger, the exact mechanisms of its participation in Ca2+ handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca2+ movements in cardiomyocytes and exposed to an NHE inhibitor, 5-( N-methyl- N-isobutyl)amiloride (MIA). [Ca2+]i in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca2+]i and augmented the KCl-induced increase in [Ca2+]i in a concentration-dependent manner. The MIA-induced increase in basal [Ca2+]i was unaffected by extracellular Ca2+, antagonists of the sarcolemmal (SL) L-type Ca2+ channel, and inhibitors of the SL Na+/Ca2+ exchanger, SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. However, the MIA-induced increase in basal [Ca2+]i was attenuated by inhibitors of SL Na+-K+-ATPase and sarcoplasmic reticulum (SR) Ca2+ transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca2+ concentration and attenuated by agents that inhibit SL L-type Ca2+ channels, the SL Na+/Ca2+ exchanger, SL Na+-K+-ATPase, and SR Ca2+ release channels and the SR Ca2+ pump. However, the effect of MIA on the KCl-induced increase in [Ca2+]i remained unaffected by treatment with inhibitors of SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca2+]i, whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca2+]i in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca2+]i and that MIA-induced increases in basal [Ca2+]i, as well as augmentation of the KCl-induced increase in [Ca2+]i, in cardiomyocytes are regulated differentially.

Intracellular calcium signals in response to bradykinin in individual neuroblastoma cells

1995 ◽  
Vol 269 (4) ◽  
pp. C841-C848 ◽  
Author(s):  
J. S. Coggan ◽  
S. H. Thompson
Keyword(s):  
Intracellular Calcium ◽  
Neuroblastoma Cells ◽  
Receptor Subtype ◽  
Half Time ◽  
Calcium Signals ◽  
Fura 2 ◽  
Low Concentrations ◽  
Intracellular Ca ◽  
High Concentrations ◽  
20 Nm

The Ca indicator fura 2 was used to study the modulation of cytoplasmic Ca by bradykinin (Bk) in single N1E-115 murine neuroblastoma cells. Increases in cytoplasmic Ca in response to Bk were mediated by the B2 receptor subtype. Responses to high concentrations of Bk (1-100 nM) were homogeneous and characterized by a rapidly rising transient that decayed to baseline in the continued presence of agonist, with a half-time of 15 s. Responses to low concentrations of Bk (100-500 pM) were more heterogeneous, with longer latencies and often with oscillations. Pretreatment with thapsigargin for 20 min prevented the Ca response, showing that the Ca change results from intracellular Ca release. Removal of external Ca had little effect on the response to Bk, indicating that the agonist does not activate Ca influx. The extent of Ca release and refilling after Bk was tested with ionomycin. A saturating dose of Bk (20 nM) mobilizes > 90% of stored Ca within 30 s, and this is replaced slowly. Replacement of external Na by N-methyl-D-glucamine to block Na/Ca exchange affected the Ca response, causing decreases in latency and in the period of Ca oscillations and increases in overall duration and peak amplitude of the response.

scholarly journals cADP ribose and [Ca2+]iregulation in rat cardiac myocytes

2000 ◽  
Vol 279 (4) ◽  
pp. H1482-H1489 ◽  
Author(s):  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Timothy F. Walseth ◽  
Gary C. Sieck
Keyword(s):  
Electrical Stimulation ◽  
Sarcoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Real Time ◽  
Cardiac Myocytes ◽  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Adult Rat ◽  
Intracellular Ca ◽  
Rat Cardiac Myocytes

cADP ribose (cADPR)-induced intracellular Ca2+ concentration ([Ca2+]i) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1–10 μM) induced sustained, concentration-dependent [Ca2+]i responses ranging from 50 to 500 nM, which were completely inhibited by 20 μM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca2+]i waves, increasing concentrations of cADPR increased wave frequency up to ∼250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca2+]i transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca2+]i responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca2+]i responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca2+]i response. These results indicate that in cardiac myocytes, cADPR induces Ca2+ release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.

In situ SR function in postinfarction myocytes

1999 ◽  
Vol 87 (6) ◽  
pp. 2143-2150 ◽  
Author(s):  
Xue-Qian Zhang ◽  
Yuk-Chow Ng ◽  
Russell L. Moore ◽  
Timothy I. Musch ◽  
Joseph Y. Cheung
Keyword(s):  
Myocardial Infarction ◽  
Sarcoplasmic Reticulum ◽  
Half Time ◽  
Time Constants ◽  
Rat Hearts ◽  
Intracellular Ca ◽  
Rat Myocytes ◽  
Electrophysiological Technique ◽  
Empirical Constants

Previous studies have shown lower systolic intracellular Ca2+ concentrations ([Ca2+]i) and reduced sarcoplasmic reticulum (SR)-releasable Ca2+ contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca2+ entry via L-type Ca2+ channels was normal, but that via reverse Na+/Ca2+exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca2+ contents in MI myocytes, we measured SR Ca2+uptake and SR Ca2+ leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca2+ and inhibit SR Ca2+ uptake resulted in a 10-fold prolongation of half-time ( t ½) of [Ca2+]itransient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca2+]itransient during a twitch in rat myocytes was primarily mediated by SR Ca2+-ATPase and that the t ½ of [Ca2+]idecline is a measure of SR Ca2+uptake in situ. At 5.0 mM extracellular Ca2+, systolic [Ca2+]iwas significantly ( P ≤ 0.05) lower (337 ± 11 and 416 ± 18 nM in MI and sham, respectively) and t ½ of [Ca2+]idecline was significantly longer (0.306 ± 0.014 and 0.258 ± 0.014 s in MI and sham, respectively) in MI myocytes. The 19% prolongation of t ½ of [Ca2+]i decline was associated with a 23% reduction in SR Ca2+-ATPase expression (detected by immunoblotting) in MI myocytes. SR Ca2+ leak was measured by a novel electrophysiological technique that did not require assigning empirical constants for intracellular Ca2+buffering. SR Ca2+ leak rate was not different between sham and MI myocytes: the time constants of SR Ca2+ loss after thapsigargin were 290 and 268 s, respectively. We conclude that, independent of decreased SR filling by Ca2+ influx, the lower SR Ca2+ content in MI myocytes was due to reduced SR Ca2+ uptake and SR Ca2+-ATPase expression, but not to enhanced SR Ca2+ leak.

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