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.

Effects of sarcoplasmic reticulum Ca2+ load on the gain function of Ca2+ release by Ca2+ current in cardiac cells

1995 ◽  
Vol 268 (2) ◽  
pp. H916-H920 ◽  
Author(s):  
A. M. Janczewski ◽  
H. A. Spurgeon ◽  
M. D. Stern ◽  
E. G. Lakatta
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Cardiac Cells ◽  
Gain Function ◽  
Whole Cell ◽  
Rat Ventricular Myocytes

We studied the effects of variable sarcoplasmic reticulum (SR) Ca2+ loading on changes in the gain index of Ca2+ release from the SR, measured as the ratio of the amount of Ca2+ released to the magnitude of the Ca2+ current (ICa) integrated for the initial 20 ms of the depolarization, in whole cell voltage-clamped rat ventricular myocytes dialyzed with the Ca2+ indicator indo 1 salt at 23 degrees C. Changes in ICa were measured directly, and changes in the SR Ca2+ release were indexed by changes in the amplitudes and rates of rise of cytosolic Ca2+ (Ca2+i) transients. The SR Ca2+ load was graded by the duration of conditioning voltage-clamp steps and verified by caffeine-dependent Ca2+i transients. A train of abbreviated (from 100 to 20 ms) voltage-clamp depolarizations, which triggers SR Ca2+ release but fails to replenish the SR with Ca2+, diminished the SR Ca2+ load by 56 +/- 5%, did not alter peak ICa but reduced the amplitudes of the ICa-dependent Ca2+i transients by 52 +/- 3%, and decreased the gain index by 60 +/- 3% (SE; n = 5 or 6). Changes in the amplitudes of Ca2+i transients elicited by ICa and changes in the gain index were linearly correlated (r2 = 0.83 and 0.79, respectively; P < 0.001 for each) with changes in amplitudes of Ca2+i transients elicited by caffeine pulses applied in lieu of the respective voltage-clamp pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

1998 ◽  
Vol 76 (9) ◽  
pp. 873-881 ◽  
Author(s):  
Yasuhiro Katsube ◽  
Hisashi Yokoshiki ◽  
Lam Nguyen ◽  
Masao Yamamoto ◽  
Nicholas Sperelakis
Keyword(s):  
Steady State ◽  
Voltage Clamp ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Fast Component ◽  
Whole Cell ◽  
Single Channel Recordings ◽  
Mode 2 ◽  
Adult Cells

Postnatal changes in the slow Ca2+ current (ICa(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean ± SEM) averaged 23.2 ± 0.5 pF in neonates (n = 163) and 140 ± 4.1 pF in adults (n = 143). ICa(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7 ± 0.2 pA/pF in neonatal and 7.8 ± 0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7 ± 1.4 ms) than in the adult (6.6 ± 0.4 ms) cells (p < 0.05). Ryanodine (10 muM) significantly increased this value to 18.0 ± 1.9 in neonate (n = 8) and to 17.7 ± 2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces / 11 cells), but not in the adult cells (400 traces / 4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.Key words: Ca2+ channels; neonatal heart cells; whole-cell voltage clamp; single-channel recordings; long openings of Ca2+ channels.

Developmental change in fast Na channel properties in embryonic chick ventricular heart cells

1995 ◽  
Vol 73 (10) ◽  
pp. 1475-1484 ◽  
Author(s):  
Hideaki Sada ◽  
Takashi Ban ◽  
Takeshi Fujita ◽  
Yoshio Ebina ◽  
Nicholas Sperelakis
Keyword(s):  
Steady State ◽  
Voltage Clamp ◽  
Time Constant ◽  
Voltage Dependence ◽  
Cell Voltage ◽  
Developmental Changes ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp

To assess developmental changes in kinetic properties of the cardiac sodium current, whole-cell voltage-clamp experiments were conducted using 3-, 10-, and 17-day-old embryonic chick ventricular heart cells. Experimental data were quantified according to the Hodgkin–Huxley model. While the Na current density, as examined by the maximal conductance, drastically increased (six- to seven-fold) with development, other current–voltage parameters remained unchanged. Whereas the activation time constant and the steady-state activation characteristics were comparable among the three age groups, the voltage dependence of the inactivation time constant and the steady-state inactivation underwent a shift in the voltage dependence toward negative potentials during embryonic development. Consequently, the steady-state (window current) conductance, which was sufficient to induce automatic activity in the young embryos, was progressively reduced with age.Key words: cardiac electrophysiology, whole-cell voltage-clamp experiments, fast Na currents, heart, development, developmental changes.

Whole-cell voltage-clamp study of the fading of GABA-activated currents in acutely dissociated hippocampal neurons

1986 ◽  
Vol 56 (1) ◽  
pp. 1-18 ◽  
Author(s):  
J. R. Huguenard ◽  
B. E. Alger
Keyword(s):  
Steady State ◽  
Voltage Clamp ◽  
Time Course ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Membrane Properties ◽  
Equilibrium Potential ◽  
Spinal Cord Neurons ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp

The lability of the responses of mammalian central neurons to gamma-aminobutyric acid (GABA) was studied using neurons acutely dissociated from the CA1 region of the adult guinea pig hippocampus as a model system. GABA was applied to the neuronal somata by pressure ejection and the resulting current (IGABA) recorded under whole-cell voltage clamp. In initial experiments we examined several basic properties of cells in this preparation. Our data confirm that passive and active membrane properties are similar to those which characterize cells in other preparations. In addition, GABA-dependent conductance (gGABA), reversal potential (EGABA), and the interaction of GABA with pentobarbital and bicuculline all appeared to be normal. Dendritic GABA application could cause depolarizing GABA responses, and somatic GABA application caused hyperpolarizations due to chloride (Cl-) movements. Repetitive brief applications (5-15 ms) of GABA (10(-5) to 10(-3) M) at a frequency of 0.5 Hz led to fading of successive peaks of IGABA until, at a given holding potential, a steady state was reached in which IGABA no longer changed. Imposing voltage steps lasting seconds during a train of steady-state GABA responses led initially to increased IGABA that then diminished with maintenance of the step voltage. The rate of decrease of IGABA at each new holding potential was independent of the polarity of the step in holding potential but was highly dependent on the rate of GABA application. Application rates as low as 0.05 Hz led to fading of IGABA, even with activation of relatively small conductances (5-15 nS). Since IGABA evoked by somatic GABA application in these cells is carried by Cl-, the Cl- equilibrium potential (ECl) is equal to the reversal potential for IGABA, i.e., to EGABA. The fading of IGABA with changes in holding potential can be almost entirely accounted for by a shift in ECl resulting from transmembrane flux of Cl- through the GABA-activated conductance. Maneuvers that prevent changes in the intracellular concentration of Cl-ions, [Cl-]i, including holding the membrane potential at EGABA during repetitive GABA application or buffering [Cl-]i with high pipette [Cl-], prevent changes in EGABA. Desensitization of the GABA response (an actual decrease in gGABA) occurs in these neurons during prolonged application of GABA (greater than 1 s) but with a slower time course than changes in EGABA. Whole-cell voltage-clamp techniques applied to tissue-cultured spinal cord neurons indicated that rapid shifts in EGABA result from repetitive GABA application in these cells as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage and beat dependence of Ca2+ transient in feline ventricular myocytes

1989 ◽  
Vol 257 (3) ◽  
pp. H746-H759 ◽  
Author(s):  
W. H. duBell ◽  
S. R. Houser
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Rest Period ◽  
Voltage Dependent ◽  
Early Peak ◽  
Comparable Magnitude ◽  
Voltage Relationship

The positive contractile staircase after a period of rest is attributable to a positive staircase in the magnitude of the Ca2+ transient. The present study used voltage-clamp techniques and the fluorescent Ca2+ indicator, indo-1, to examine the effects of membrane potential, the duration of depolarization, and the slow inward Ca2+ current (Isi) in the regulation of the magnitude of the steady-state Ca2+ transient and the development of the steady state during the positive staircase. In the steady state, the Ca2+ transient was greatest at +10 mV, the potential at which Isi was also the greatest. However, the Isi-voltage relationship was much more bell-shaped than the Ca2+ transient-voltage relationship. The magnitude and duration of the steady-state Ca2+ transient was not affected by pulse durations as short as 25 ms. However, prolonged voltage pulses were essential to maintain the steady state. The development of the positive staircase was very voltage dependent. After a rest period, a positive staircase was seen when voltage-clamp drives were done to +30 mV but not when done to -10 mV, potentials that elicit Isi of comparable magnitude. These results support the idea that the early peak of Isi can act as a trigger for release of Ca2+ from the sarcoplasmic reticulum. However, sarcoplasmic reticulum Ca2+ loading is dependent on prolonged depolarization and may be mediated through Na+-Ca2+ exchange.

Effects of endothelin 1 on calcium and sodium currents in isolated human cardiac myocytes

1995 ◽  
Vol 73 (12) ◽  
pp. 1774-1783 ◽  
Author(s):  
Tzu-Hurng Cheng ◽  
Chung-Yi Chang ◽  
Jeng Wei ◽  
Cheng-I Lin
Keyword(s):  
Voltage Clamp ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Cardiac Cells ◽  
Sodium Currents ◽  
Atrial Myocytes ◽  
Whole Cell ◽  
Voltage Clamp Technique ◽  
Endothelin 1

We have used the whole-cell voltage-clamp technique to study the effects of endothelin 1 (ET-1, 10 nM) on L-type Ca2+ currents and voltage-dependent Na+ inward currents in human cardiac cells. Myocytes were enzymatically isolated from atrial specimens obtained during open-heart surgery and from human ventricular tissues of explanted hearts. Extracellular application of ET-1 decreased the peak amplitude of Ca2+ currents by 26 ± 6% (n = 13) in atrial myocytes and by 19 ± 3% (n = 8) in ventricular myocytes. In three atrial cells, treatment with 1 μM BQ123 prevented the decrease in Ca2+ currents induced by ET-1. When GTP (0.2 mM) was added to the dialyzing pipette solution, ET-1 still caused a small decline by 12 ± 5% (n = 16), in peak Ca2+ currents, in atrial myocytes. When Ca2+ currents were increased (+210 ± 19%) by a β-adrenoceptor agonist (0.1 μM isoproterenol) or by the phosphodiesterase inhibitor isobutylmethylxanthine (10 μM), ET-1 reduced Ca2+ currents by 35 ± 6% (n = 4) and 30 ± 4% (n = 5), respectively. In human ventricular myocytes in the presence of 1 μM isoproterenol, which increased the peak Ca2+ currents by 150 ± 30%, ET-1 also induced a drastic reduction in Ca2+ currents, by 40 ± 11% (n = 5). The tetrodotoxin-sensitive Na+ currents measured in the presence of 5 mM [Na]o were significantly enhanced (+28 ± 7%) by ET-1 in five atrial myocytes. The stimulatory effect of ET-1 on Na+ currents was partially reversible. The present findings in human cardiac cells show that ET-1 did not enhance the Ca2+ currents in the absence or presence of internal GTP. The positive inotropic actions induced by ET-1 in human heart may be mediated mainly by signal-transduction pathways other than the G-protein – adenylyl cyclase – cAMP system.Key words: endothelin 1, human cardiac myocytes, whole-cell voltage-clamp technique, calcium currents, sodium currents.

scholarly journals Low-voltage triggering of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle cells

2003 ◽  
Vol 285 (6) ◽  
pp. C1544-C1552 ◽  
Author(s):  
Fabien Brette ◽  
Jean-Yves Le Guennec ◽  
Ian Findlay
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Guinea Pig ◽  
Cardiac Muscle ◽  
Small Amplitude ◽  
Ventricular Myocytes ◽  
Low Voltage ◽  
Cell Voltage ◽  
Slip Mode ◽  
Cardiac Muscle Cells ◽  
Whole Cell

This study investigated the interaction between L-type Ca2+ current (ICaL) and Ca2+ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Nao+:KI+) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca2+ current, TTX-sensitive ICa (ICaTTX), or “slip mode” Ca2+ conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca2+ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca2+ release from the SR at negative potentials is due to activation of L-type Ca2+ channels.

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.

Reexamination of the Stoichiometry of Na+/Ca2+ Exchange with Whole-Cell Voltage Clamp of Guinea Pig Ventricular Myocytes

2006 ◽  
Vol 976 (1) ◽  
pp. 154-156 ◽  
Author(s):  
MASAMITSU HINATA ◽  
HISAO YAMAMURA ◽  
LIBING LI ◽  
YASUHIDE WATANABE ◽  
TOMOKAZU WATANO ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp
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