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)

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.

ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+

1989 ◽  
Vol 257 (4) ◽  
pp. C750-C758 ◽  
Author(s):  
M. B. De Young ◽  
A. Scarpa
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ethylene Glycol ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Channel Blockers ◽  
Tetraacetic Acid ◽  
Free Medium ◽  
Fura 2 ◽  
Adult Rat ◽  
Ca2 Channels

Addition of micromolar concentrations of extracellular ATP to adult rat cardiac ventricular myocytes increases cytosolic Ca2+ concentration ([Ca2+]). Experiments were performed on fura-2-loaded myocytes to determine whether the [Ca2+] rise was due to Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum (SR), or a combination of both. BAY K 8644 and nifedipine affected ATP-induced [Ca2+] transients, indicating involvement of voltage-sensitive Ca2+ channels. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+ channel blockers significantly reduced cytosolic [Ca2+] changes due to addition of ATP or KCl without depleting Ca2+ stores (shown by ionomycin treatment in a Ca2+-free medium), demonstrating that these responses require Ca2+ influx. Depletion of intracellular Ca2+ stores by caffeine or ryanodine also diminished cytosolic [Ca2+] responses, indicating that a portion of the increased cytosolic [Ca2+] is due to Ca2+ release from SR. Norepinephrine potentiates the ATP-Ca2+ response, and this effect was not inhibited by depletion of intracellular stores. Although the data show that there are two Ca2+ sources in the cytosolic Ca2+ response to ATP, the pattern is also consistent with the hypothesis of Ca2+-induced Ca2+ release from cardiac SR.

scholarly journals Sarcolemmal distribution of ICa and INCX and Ca2+ autoregulation in mouse ventricular myocytes

2017 ◽  
Vol 313 (1) ◽  
pp. H190-H199 ◽  
Author(s):  
Hanne C. Gadeberg ◽  
Cherrie H. T. Kong ◽  
Simon M. Bryant ◽  
Andrew F. James ◽  
Clive H. Orchard
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Surface Membrane ◽  
Ryanodine Receptors ◽  
Cell Voltage ◽  
Intact Cells ◽  
T Tubules

The balance of Ca2+ influx and efflux regulates the Ca2+ load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca2+ influx, via L-type Ca2+ current ( ICa), and efflux, via the Na+/Ca2+ exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of ICa and NCX current ( INCX), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca2+ measurements in intact and detubulated (DT) cells. In contrast to the rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca2+ observed in intact myocytes was preserved after detubulation. Immunostaining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of the Ca2+ transient amplitude to steady state (autoregulation) after application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However, during application of 200 µM caffeine to increase sarcoplasmic reticulum (SR) Ca2+ release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca2+ release is a consequence of the distribution of ICa, which is reduced in DT cells and is required to refill the SR after depletion, and NCX, which is little affected by detubulation, remaining available to remove Ca2+ when SR Ca2+ release is increased. NEW & NOTEWORTHY This study shows that in contrast to the rat, mouse ventricular Na+/Ca2+ exchange current density is lower in the t-tubules than in the surface sarcolemma and Ca2+ current is predominantly located in the t-tubules. As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release.

Protein kinase C-ζ modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt

2002 ◽  
Vol 282 (1) ◽  
pp. H320-H327 ◽  
Author(s):  
Yukitaka Shizukuda ◽  
Peter M. Buttrick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adult Rat ◽  
Kinase C ◽  
Adult Cardiac Myocytes ◽  
Pkc Ζ

We hypothesized that thromboxane A2 (TxA2) receptor stimulation directly induces apoptosis in adult cardiac myocytes. To investigate this, we exposed cultured adult rat ventricular myocytes (ARVM) to a TxA2 mimetic [1S-[1α,2α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) for 24 h. Stimulation with I-BOP induced apoptosis in a dose-dependent manner and was completely prevented by a TxA2 receptor antagonist, SQ-29548. We further investigated the role of protein kinase C (PKC) in this process. TxA2 stimulation resulted in membrane translocation of PKC-ζ but not PKC-α, -βII, -δ, and -ε at 3 min and 1 h. The activation of PKC-ζ by I-BOP was confirmed using an immune complex kinase assay. Treatment of ARVM with a cell-permeable PKC-ζ pseudosubstrate peptide (ζ-PS) significantly attenuated apoptosis by I-BOP. In addition, I-BOP treatment decreased baseline Akt activity and its decrease was reversed by treatment with ζ-PS. The inhibition of phosphatidylinositol 3-kinase upstream of Akt by wortmannin or LY-294002 abolished the antiapoptotic effect of ζ-PS. Therefore, our results suggest that the activation of PKC-ζ modulates TxA2 receptor-mediated apoptosis at least, in part, through Akt activity in adult cardiac myocytes.

Steady-state twitch Ca2+ fluxes and cytosolic Ca2+ buffering in rabbit ventricular myocytes

1996 ◽  
Vol 270 (1) ◽  
pp. C192-C199 ◽  
Author(s):  
L. M. Delbridge ◽  
J. W. Bassani ◽  
D. M. Bers
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Whole Cell ◽  
Rabbit Ventricular Myocytes ◽  
The Mean ◽  
State Contraction ◽  
Caffeine Contractures

Intracellular Ca2+ ([Ca2+]i) transients and transsarcolemmal Ca2+ currents were measured in indo 1-loaded isolated rabbit ventricular myocytes during whole cell voltage clamp to quantitate the components of cytosolic Ca2+ influx and to describe the dynamic aspects of cytosolic Ca2+ buffering during steady-state contraction (0.5 Hz, 22 degrees C). Sarcolemmal Ca2+ influx was directly measured from the integrated Ca2+ current (Ica) recorded during the clamp (158 +/- 10 attomoles; amol). Sarcoplasmic reticulum (SR) Ca2+ content was determined from the integrated electrogenic Na+/Ca2+ exchange current (Ix) induced during rapid application and sustained exposure of cells to caffeine to elicit the release of the SR Ca2+ load (1,208 +/- 170 amol). The mean steady-state SR Ca2+ load was calculated to be 87 +/- 13 microM (mumol/l nonmitochondrial cytosolic volume). Ca2+ influx via Ica represented approximately 14% of the stored SR Ca2+ and 23% of the total cytosolic Ca2+ flux during a twitch (47 +/- 6 microM). Comparison of electrophysiologically measured Ca2+ fluxes with Ca2+ transients yields apparent buffering values of 60 for caffeine contractures and 110 for twitches (delta Ca2+ total/delta Ca2+ free). This is consistent with the occurrence of "active" buffering of cytosolic Ca2+ by SR Ca2+ uptake during the twitch.

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.

Voltage-independent changes in L-type Ca2+current uncoupled from SR Ca2+ release in cardiac myocytes

2000 ◽  
Vol 279 (4) ◽  
pp. H2024-H2031 ◽  
Author(s):  
Andrzej M. Janczewski ◽  
Edward G. Lakatta ◽  
Michael D. Stern
Keyword(s):  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Abrupt Increase ◽  
Time To Peak ◽  
Time Integral ◽  
Marked Disparity ◽  
Release Flux ◽  
Intracellular Ca ◽  
Combined Actions ◽  
Continuous Presence

To determine the effect of voltage-independent alterations of L-type Ca2+ current ( I Ca) on the sarcoplasmic reticular (SR) Ca2+ release in cardiac myocytes, we measured I Ca and cytosolic Ca2+ transients (Cai 2+; intracellular Ca2+ concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca2+] (Cao 2+) or 2) application of 1 μM FPL-64176, a Ca2+channel agonist, to selectively alter I Ca in the absence of changes in SR Ca2+ loading. On the first depolarization in higher Cao 2+, peak I Ca was increased by 46 ± 6% ( P < 0.001), but the increases in the maximal rate of rise of Cai 2+(dCai 2+/d t max, where t is time; an index of SR Ca2+ release flux) and the Cai 2+ transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I Ca, increasing its time integral by 117 ± 8% ( P < 0.001) without significantly increasing peak I Ca, dCai 2+/d t max, or amplitude of the corresponding Cai 2+ transient. Prolongation of exposure to higher Cao 2+ or FPL-64176 did not further increase peak I Ca but greatly increased dCai 2+/d t max, Cai 2+ transient amplitude, and the gain of Ca2+ release (dCai 2+/d t max/ I Ca), evidently due to augmentation of the SR Ca2+ loading. Also, the time to peak dCai 2+/d t maxwas significantly increased in the continuous presence of higher Cao 2+ (by 37 ± 5%, P < 0.001) or FPL-64176 (by 63 ± 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I Ca and the corresponding SR Ca2+ release. We attribute this to saturation of the SR Ca2+ release flux as predicted by local control theory. Prolongation of the SR Ca2+ release flux, caused by combined actions of a larger I Ca and maximally augmented SR Ca2+ loading, might reflect additional Ca2+ release from corbular SR.

Effects of Saponin Monomer 13 of Dwarf Lilyturf Tuber on L-Type Calcium Currents in Adult Rat Ventricular Myocytes

2005 ◽  
Vol 33 (05) ◽  
pp. 797-806 ◽  
Author(s):  
Jin Tao ◽  
Hongyi Wang ◽  
Jiandong Chen ◽  
Huae Xu ◽  
Shengnan Li
Keyword(s):  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Inhibitory Effects ◽  
Patch Clamp Recording ◽  
Inactivation Curve ◽  
Control Value ◽  
Adult Rat ◽  
Cardioprotective Effects ◽  
Rat Cardiac Myocytes

The saponin monomer 13 of dwarf lilyturf tuber (DT-13), one of the saponin monomers of dwarf lilyturf tuber, has been found to have potent cardioprotective effects. In order to investigate the effect of DT-13 on L-type calcium currents ( I Ca,L ), exploring the mechanisms of DT-13's cardioprotective effects, we directly measured the I Ca,L in the adult rat cardiac myocytes exposed to DT-13 using standard whole-cell patch-clamp recording technique. Our results showed that DT-13 exerted inhibitory effects on the I Ca,L of the single adult rat cardiac myocytes. The current density was reduced by about 38% after exposure of the cells to DT-13 (0.1 μM) for 10 minutes, from the control value of 7.46 ± 1.31 pA/pF to 4.25 ± 0.35 pA/pF ( n = 6, p < 0.05). This I Ca,L -inhibiting action of DT-13 was concentration-dependent. DT-13 up-shifted the current-voltage (I-V) curve, but did not significantly affect the half activation potential (V0.5). V0.5 was from -11.8 ± 0.9 mV in the control to -12.6 ± 1.9 mV in the presence of DT-13 at 0.1 μmol/L. DT-13 at 0.1 μM did not markedly affect the activation of I Ca,L , but shifted the inactivation curve of I Ca,L to the left. In combination with previous reports, these results suggest that there might be a close relationship between the cardioprotective effects of dwarf lilyturf tuber and the inhibitory effects of DT-13 on L-type calcium currents.

Hyperthyroid adult rat cardiomyocytes. II. Single cell electrophysiology and free calcium transients

1989 ◽  
Vol 257 (5) ◽  
pp. C957-C963 ◽  
Author(s):  
Q. Li ◽  
Z. Guan ◽  
B. A. Biagi ◽  
B. T. Stokes ◽  
R. A. Altschuld
Keyword(s):  
Single Cell ◽  
Action Potentials ◽  
Mechanical Performance ◽  
Ventricular Myocytes ◽  
Calcium Transients ◽  
Calcium Handling ◽  
Free Calcium ◽  
Half Time ◽  
Rat Cardiomyocytes ◽  
Adult Rat

The effects of hyperthyroidism on electrophysiological properties and intracellular free calcium transients in single adult rat cardiomyocytes were studied using conventional microelectrodes and time-resolved single cell fura-2 fluorescence microscopy. Under control conditions, resting membrane potentials and triggered action potentials were not different in euthyroid and hyperthyroid myocytes. Calcium transients produced by electrical stimulation, however, were markedly abbreviated in hyperthyroid myocytes. During a train of stimuli, the duration of the calcium transients at half peak amplitude (half time) was 124 +/- 14 ms at the fifth beat in hyperthyroid cells vs. 287 +/- 35 ms in euthyroid cells. Isoproterenol (1 microM) prolonged time to 50% repolarization (APD50) of the action potentials and increased the peak calcium transients in both euthyroid and hyperthyroid myocytes. It also shortened the half time of the calcium transients in euthyroid myocytes but had little effect on the half time in hyperthyroid cells. These data are consistent with the electrophysiology and mechanical performance in intact euthyroid and hyperthyroid cardiac tissues, and the intrinsic changes in hyperthyroid tissues can therefore be illustrated in single ventricular myocytes. Furthermore, the results suggest that alterations in intracellular calcium handling by sarcoplasmic reticulum may account for contractile changes of the heart induced by hyperthyroidism.

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