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.
In many mammalian tissue types an integral membrane protein--the sodium/calcium (Na/Ca) exchanger--plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig 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 (INaCa) to be measured during a descending voltage ramp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. The application of 10 mM Ni caused a maximal block of INaCa since inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCa were assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 microM cAMP-containing solutions. At +60 mV, Ni inhibited INaCa in cells dialysed with a cAMP-free solution with a dissociation constant (KD) 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 microM cAMP the exchange was inhibited by Ni with a KD of 0.16 +/- 0.05 mM, the Hill coefficient was 0.82 +/- 0.16 (n = 6-7 cells). The KD and Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCa by Ni did not appear to be voltage-dependent, was maximal within 3-4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was 'mixed' showing competition with external Ca and a degree of non-competitive block. With 100 microM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCa in guinea-pig cardiac cells.
Hinde
A K
AK
Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, UK. A.K.HINDE@bristol.ac.uk
Perchenet
L
L
Hobai
I A
IA
Levi
A J
AJ
Hancox
J C
JC
D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D015314:Dialysis Solutions; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors*; D013997:Time Factors
D015314:Dialysis Solutions; D019831:Sodium-Calcium Exchanger; D009532:Nickel; D000242:Cyclic AMP