Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells.
1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use of Sylgard-coated patch pipettes (3-6 M[omega]). High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier controlled by the 'E9SCREEN' software. In addition, a charting programme on another computer synchronously recorded at low resolution (2 Hz) membrane potential and holding current (low-pass filtered at 500 Hz). 3. Na+/K+-ATPase activity was measured as the ouabain-sensitive change in the zero-current potential. The zero-current potential in rat peritoneal mast cells measured 2 min after obtaining whole-cell configuration amounted to 1.7 +/- 2.5 mV (n = 21). Ouabain (5 mM), a Na+/K+-ATPase-inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n = 3). 4. When mast cells were superfused with nominal calcium-free external solution, the cells hyperpolarized (delta mV: 20.2 +/- 3.8 mV (n = 5)). In addition, when the mast cells were preincubated in nominal calcium-free external solution for 12 +/- 1.6 min before whole-cell configuration, the membrane potential amounted to -53.7 +/- 9.8 mV (n = 8). A subsequent superfusion with ouabain (5 mM) depolarized the membrane potential (ouabain-sensitive hyperpolarization (delta mV): 23.0 +/- 8.4 mV (n = 8)). 5. A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (delta mV: 20.2 +/- 9.1 mV (n = 7)), but only when ATP was present. A subsequent superfusion with ouabain (5 mM) repolarized these cells to -1.2 +/- 14 mV (ouabain-sensitive hyperpolarization (delta mV): 19.7 +/- 7.7 mV (n = 7)). 6. The size of the [Na+]i-dependent hyperpolarization was dose-dependent. Low [Na+]i (1 mM) had no effect on membrane potential and these cells were unaffected by superfusion with calcium-free external solution. 7. These data thus directly confirm that the stimulant effect of calcium-free external solutions on the ouabain-sensitive changes in the zero-current potential, and hence the Na+/K+-ATPase, is mediated through [Na+]i and that the activity of the Na+/K+-ATPase can have an important influence on the resting membrane potential in rat peritoneal mast cells.
Friis
U G
UG
Department of Pharmacology, Odense University, Denmark.
Praetorius
H A
HA
Knudsen
T
T
Johansen
T
T
D000818:Animals; D002118:Calcium / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010529:Peritoneal Cavity / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology*
D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium