Intracellular calcium and Na+-Ca2+ exchange current in isolated toad pacemaker cells

Abstract

1. Single pacemaker cells were isolated from the sinus venosus of cane toad (Bufo marinus) in order to study the mechanisms involved in the spontaneous firing rate of action potentials. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 to determine whether [Ca2+]i influenced firing rate. A rapid transient rise of [Ca2+]i was recorded together with each spontaneous action potential. [Ca2+]i at the peak of systole was 655 +/- 64 nM and the minimum at the end of diastole was 195 +/- 15 nM. 2. Reduction of extracellular Ca2+ concentration from 2 to 0.5 mM caused a reduction in both systolic and diastolic [Ca2+]i and the spontaneous firing rate also gradually declined. 3. Application of the acetoxymethyl (AM) ester of BAPTA (10 microM), in order to increase intracellular calcium buffering, caused a decline in systolic and diastolic [Ca2+]i. The firing rate declined progressively until the cells stopped firing after 10-15 min. At the time that firing ceased, the diastolic [Ca2+]i had declined by 141 +/- 38 nM. 4. In the presence of ryanodine (2 microM), which interferes with Ca2+ release from the sarcoplasmic reticulum, the systolic and diastolic [Ca2+]i both declined and the firing rate decreased until the cells stopped firing. At quiescence diastolic [Ca2+]i had declined by 93 +/- 20 nM. 5. Exposure of the cells to Na+-free solution caused a rise in [Ca2+]i which exceeded the systolic level after 4.8 +/- 0.3 s. This rise is consistent with Ca2+ entry on a Na+-Ca2+ exchanger. 6. Rapid application of caffeine (10-20 mM) to cells clamped at -60 mV caused a rapid increase in [Ca2+]i which then spontaneously declined. An inward current with a similar time course to that of [Ca2+]i was also generated. Application of Ni2+ (5 mM) or 2,4-dichlorobenzamil (25 microM) reduced the amplitude of the inward current produced by caffeine by 96 +/- 1 % and 74 +/- 10 %, respectively. In a Na+-free solution the caffeine-induced current was reduced by 93 +/- 7 %. 7. Under a variety of circumstances the diastolic [Ca2+]i showed a close association with pacemaker firing rate. The existence of a Na+-Ca2+ exchanger and its estimated contribution to inward current during the pacemaker potential suggest that the Na+-Ca2+ exchange current makes a contribution to pacemaker activity.

Figure 1. The calibration of intracellular indo-1

A, the whole-cell patch clamp technique was used to buffer the cell [Ca²⁺] to various levels set by Ca²⁺-EGTA buffers in the patch pipette. The pipette also contained 400 μm indo-1. •, means ± s.e.m of experimental data from at least 5 cells for each point (error bars in most cases smaller than symbols). ▵, ratios achieved with EGTA alone (R_min) and Ca²⁺+ CaEGTA (R_max). The continuous line is the least squares fit of eqn (1) to the data points and gave a K_dβ= 1720 ± 130 nm. B, the decline of fluorescence at 400 and 510 nm (F₄₀₀ and F₅₁₀) after breaking the membrane of a cell loaded with indo-1 AM. The labels in the figure represented background (a); the F₄₀₀ and F₅₁₀ from an indo-1 AM loaded cell before (b) and after breaking the cell membrane (c). F₅₁₀ signal was shifted down by 0.5 unit to help distinguish the two traces.

Figure 2. Simultaneously recorded action potentials and [Ca²⁺]_i signals from a spontaneously firing toad pacemaker cell

A, simultaneously recorded action potentials (upper panel) and calcium transients (lower panel). Action potentials were recorded using the nystatin perforated-patch technique and have been corrected for the calculated liquid junction potentials (−5 mV). B, superimposed action potential and [Ca²⁺]_i from a different cell from A. The action potential data has been smoothed with 10 Hz low-pass filter to match the filtering of the [Ca²⁺]_i signal.

Figure 3. The effect of low [Ca²⁺]_o on the [Ca²⁺]_i and firing rate of a toad pacemaker cell

A, continuous record of [Ca²⁺]_i (lower panel) during exposure to reduced extracellular Ca²⁺ concentration (reduced from 2 to 0.5 mm). Upper panel shows the instantaneous, spontaneous firing rate calculated from the interval between each Ca²⁺ transient. B and C, the correlation between firing rate and diastolic [Ca²⁺]_i (B) and systolic [Ca²⁺]_i (C). The symbols represent different experiments (n= 5). The values of correlation coefficient (r) are shown on each panel and are statistically significant (P < 0.05).

Figure 4. The effect of exposure to BAPTA AM on the [Ca²⁺]_i and the spontaneous action potential recorded from different toad pacemaker cells

A, [Ca²⁺]_i recorded under control and after 5 and 8 min exposure to 10 μm BAPTA AM. B, the effects of BAPTA AM on spontaneous action potential were recorded from a non-indo loaded cell. The panels show control, 5 and 8 min exposure. C and D, the correlations between firing rate and [Ca²⁺]_i from 12 measurements of 5 cells. C shows diastolic [Ca²⁺]_i and D shows systolic [Ca²⁺]_i. The correlation coefficients are shown (r) on each panel and are statistically significant (P < 0.01).

Figure 5. The effects of 10 μm of ryanodine on the [Ca²⁺]_i and the spontaneous action potential from different toad pacemaker cells

A, [Ca²⁺]_i recorded under control and after 5 and 30 min exposure to ryanodine (10 μm). B, the effects of ryanodine on spontaneous action potentials were recorded from a non-indo loaded cell. The panels show control and 5 and 30 min exposure. C and D, the correlations between firing rate and [Ca²⁺]_i from 14 measurements of 8 cells. C shows diastolic [Ca²⁺]_i and D shows systolic [Ca²⁺]_i. The correlation coefficients are shown (r) on each panel and are significant (P < 0.01).

Figure 6. The effect of Na⁺-free solution on [Ca²⁺]_i recorded from a toad pacemaker cell

Extracellular Na⁺ was replaced by N-methyl glucamine. A, application of Na⁺-free solution abolishes spontaneous firing and causes a rapid increase in [Ca²⁺]_i. B, superimposed records of [Ca²⁺]_i on exposure to Na⁺-free solution from the same cell before and after 30 min exposure to 2 μm ryanodine.

Figure 7. Inward current and [Ca²⁺]_i induced by rapid application of 10 mm caffeine

The nystatin perforated-patch technique was used to record the whole-cell current. A Cs⁺-rich solution was used in the pipette to block K⁺ currents as described in the method section. The cell was voltage clamped at −60 mV. A, rapid application of 10 mm caffeine induced an inward current (upper panel) with the time course similar to that of [Ca²⁺]_i (lower panel). Line drawn through declining phase of [Ca²⁺]_i is an exponential fit whose time constant (τ) is shown. B, caffeine and 5 mm Ni²⁺ applied simultaneously. The inward current was largely blocked while the [Ca²⁺]_i increase was larger but declined more slowly. Exponential fit to early [Ca²⁺]_i decline is shown by line and time constant (τ).

Figure 9. Relations between the caffeine-induced inward current and the [Ca²⁺]_i

A, time course of caffeine-induced current (continuous line, inward current plotted upward); dashed line shows the [Ca²⁺]_i signal. Both signals filtered with a 10 Hz low-pass filter. B, plot of peak caffeine-induced current and peak [Ca²⁺]_i. Ca²⁺ release was varied by repeating caffeine exposures before the SR had time to fully reaccumulate Ca²⁺. Data from 3 cells shown each as different symbols. Continuous line show a linear regression of these points. C, plot of the caffeine-induced inward current versus[Ca²⁺]_i throughout a single caffeine exposure. Arrow indicates the rising phase of current and [Ca²⁺]_i. The dotted line represents the calculated Na⁺-Ca²⁺ exchange current using the model of Rasmussen et al. (1990) and is given by the equation: where K_Na,Ca is a scaling factor (4 × 10⁻⁶) which represents the magnitude of the current for a given set of ionic gradients, V is the membrane potential (−60 mV), concentrations are all in millimolar and I_Na,Ca is in nanoamps for a representative bull frog pacemaker cell. [Na⁺]_i was set to 10 mm (the concentration in the pipette solution) and [Na⁺]_o and [Ca²⁺]_o were set to the bath solution (115 and 2 mm respectively). •, shows the mean and s.e.m. for the peak of the caffeine-induced current and the peak of the caffeine-induced [Ca²⁺]_i signal from 12 cells. The dashed line shows the modified Rassmussen model fitted to the mean of the 12 data points which required an 11-fold increase in the value of K_Na,Ca.

Figure 8. Evidence that the inward current induced by caffeine is Na⁺-Ca²⁺ exchange current

An indo-1 loaded cell was voltage clamped at −60 mV. A, first application of caffeine (10 mm) induced a typical inward current and rise in [Ca²⁺]_i. Reapplication of caffeine after 20 s caused only a small [Ca²⁺]_i increase and a small inward current. B, control caffeine application on the left. Right hand panel shows same cell after 5 s perfusion with Na⁺- and Ca²⁺-free solution. Caffeine application under these conditions caused a greatly reduced inward current while the [Ca²⁺]_i increase was larger and declined more slowly.