Cytosolic free Ca2+ during operation of sodium‐calcium exchange in guinea‐pig heart cells.

Abstract

SUMMARY1. Membrane current generated by the Na+-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltageclamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa Ca) was measured from records of the 2 mM-Ni2+-sensitive current and used in an attempt to clamp [Ca2+ ]i at a level determined by the ionic compositions of the external and pipette solutions.2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa ca expected for a 3Na+: ICa2+ exchanger. The measured value of ENa ca was more positive than the theoretical value when the membrane potential was held positive to ENa ca and the opposite was true when the holding potential was more negative than the expected ENa Ca.3. As Indo-l diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly.4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+.5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) A. NOMA AND OTHERS increase in [Ca2+]i estimated from Indo-1 using the extracellular calibration curve, but the values of the influx determined directly from Indo-1 fluorescence were always larger than those calculated from the exchanger current.6. Hyperpolarization to -80 mV from a holding potential of -40 mV decreased[Ca2+]i, while the membrane slope conductance for outward Na+-Ca2+ exchange current increased rapidly. The opposite was true when the membrane was depolarized. These effects were reversed by clamping back to -40 mV. Membrane conductance for the inward exchanger current did not change markedly during these manoeuvres. 7. These observations suggest that when Indo-1 is applied through a dialysing patch pipette the resulting intracellular fluorescence can be calibrated to give a satisfactory estimate of [Ca2+]i, and that changes in membrane potential produce marked variations in [Ca2+]i via modulation of Na+-Ca2+ exchange.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

“…In low-Ca2+ buffered and contracting myocytes, the suppressive effect of isoproterenol was associated with a -20 mV shift in the reversal potential of INa-Ca, whereas in highly buffered, noncontracting myocytes ([Ca2+1] controlled at 200 nM), no significant change in the reversal potential of INa-Ca was found (Table 1). It should be noted that in high-Ca2+ buffer internal solutions (free [Ca2+1] = 200 nM, calculated using the CA-BUF program), the theoretically predicted reversal potential for the exchanger (ENa-Ca = 3ENa -2ECa) (33)(34)(35) was -106.7 mV and deviated significantly from the Erev of -62.8 ± 10.9 mV measured using the ramp-clamp protocol (Table 1; Fig. 4 A and B).…”

Sodium/Calcium Exchanger: Influence of Metabolic Regulation on Ion Carrier Interactions

“…These measurements of coupling ratio by employing different methods lead to the conclusion that the cardiac/ neuronal Na ϩ /Ca 2ϩ has a coupling ratio of 3Na ϩ for 1Ca 2ϩ whether the exchanger is operating in the forward or reverse mode. More recently, this subject has been revised by Fujioka et al (109), who take the view that precise measurements of the reversal potential of the exchanger are difficult as a consequence of accumulation and depletion of ions near the plasma membrane (106,194). Although this problem seems to be small in the giant excised membrane patches, the above authors argue that the uses of "bleb" membrane instead of intact cardiac sarcolemmal could alter the Na ϩ /Ca 2ϩ exchange properties (109).…”

Modulation of Intracellular Na+ and Ca2+ Induced by the Cardiac Na+-Ca2+ Exchanger in Guinea Pig Ventricular Myocytes

“…In the present study we have attempted to measure the changes in [Ca 2ϩ ] i and [Na ϩ ] i by measuring membrane current arising from exchanger turnover (I NCX ) in single guinea pig ventricular myocytes while monitoring [Ca 2ϩ ] i with the fluorescent probe Indo-1 applied through a perfused patch pipette [19]. Our results demonstrate that in external solutions containing low levels of Ca 2ϩ (ϳ66 M ] i , mainly by release from internal stores, even though the patch pipette contains a high level of Ca 2ϩ buffering, well in excess of that which abolishes phasic contractions.…”

“…The level of cytosolic free Ca 2ϩ ([Ca 2ϩ ] i ) was measured by using the fluorescent probe Indo-1 (100 M, potassium salt, Molecular Probes, Eugene, or Calbiochem, UK) loaded into single myocytes via a perfused patch pipette, and the emitted fluorescence was monitored on a modified inverted microscope (TMD, Nikon), using the same methods described in detail by Noma et al [19]. The ratio (R) of the emitted fluorescence (corrected for background) at wavelengths of 405 and 480 nm was used to calculate [Ca 2+ ] i , using the relationship…”

“…Because the optical setup was identical to the one used previously [19], with the exception that the Xenon lamp was replaced by a new unit, we followed the experimental procedure described in [19] to obtain the calibration factors of R max ϭ1.01, R min ϭ0.063, S f2 /S b2 ϭ3.5, and K d ϭ356 nM to determine [Ca 2ϩ ] i . As in [19], R min was determined intracellularly, and the remaining calibration values were determined extracellularly. In Fig.…”