Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 degrees C, dialysed internally with cAMP-free and cAMP-containing solutions
Ak. Hinde et al., Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 degrees C, dialysed internally with cAMP-free and cAMP-containing solutions, CELL CALC, 25(4), 1999, pp. 321-331
In many mammalian tissue types an integral membrane protein - the sodium/ca
lcium (Na/Ca) exchanger plays a key role in intracellular Ca homeostasis, a
nd evidence suggests that Na/Ca exchange function can be modulated by cAMP-
dependent phosphorylation. External Nickel (Ni) ions are used widely to inh
ibit the exchange but little is known about the mode of Ni action. In guine
a-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by
external Ni under phosphorylated (cells dialysed with cAMP) and non-phosph
orylated conditions. Ventricular myocytes were isolated from adult guinea-p
ig hearts, recordings were made at 37 degrees C using the whole-cell patch
clamp technique. Internal and external solutions were used which allowed Na
/Ca exchange current (I-NaCa) to be measured during a descending voltage ra
mp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. Th
e application of 10 mM Ni caused a maximal block of I-NaCa since inhibition
was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superf
used externally. Kinetics of Ni-block of I-NaCa,a,a were assessed using app
lications of different external [Ni] to cells dialysed internally with cAMP
-free and 100 mu M cAMP-containing solutions. At +60 mV, Ni inhibited I-NaC
a in cells dialysed with a cAMP-free solution with a dissociation constant
(K-D) of 0.29 +/- 0.03 mM and the data were fitted with a Hill coefficient
of 0.89 +/- 0.07 (n = 9 cells). In cells dialysed with 100 mu M cAMP the ex
change was inhibited by Ni with a K-D of 0.16 +/- 0.05 mM, the Hill coeffic
ient was 0.82 +/- 0.16 (n = 6-7 cells). The K-D and Hill coefficient values
obtained in cells dialysed with cAMP-free and cAMP-containing solutions we
re not significantly different. Inhibition of I-NaCa by Ni did not appear t
o be voltage-dependent, was maximal within 3-4 s of application and was rap
idly reversible. With cAMP-free internal dialysate, inhibition was 'mixed'
showing competition with external Ca and a degree of non-competitive block.
With 180 mu M cAMP the inhibition appeared to be more non-competitive. We
conclude that, under these experimental conditions, a concentration of exte
rnal Ni of 10 mM is sufficient to produce maximal inhibition of I-NaCa in g
uinea-pig cardiac cells.