Role of the Na+/K+‐ATPase in regulating the membrane potential in rat peritoneal mast cells

Open Access
Publisher Website
Google Scholar
Abstract
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. Experiments were performed at 22–26°C in the tight‐seal whole‐cell configuration of the patch‐clamp technique by use of Sylgard‐coated patch pipettes (3–6 MΩ). 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). 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). When mast cells were superfused with nominal calcium‐free external solution, the cells hyperpolarized (Δ 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 (Δ mV): 23.0±8.4 mV (n=8)). A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (Δ 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 (Δ mV): 19.7±7.7 mV (n=7)). 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. 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. British Journal of Pharmacology (1997) 122, 599–604; doi:10.1038/sj.bjp.0701414