The demarcation membrane system (DMS) develops in megakaryocytes (MKs) to provide additional membrane for platelet production. We recently demonstrated that whole cell capacitance measurements and confocal imaging of impermeant styryl dyes represent novel approaches to studying the DMS in living tissue (Mahaut-Smith et al. 2001). This study has now examined whether whole cell capacitance measurements reflect the entire DMS.

Male Wistar rats were humanely killed by CO₂ inhalation and cervical dislocation. Whole-cell patch clamp and confocal fluorescence recordings from MKs were conducted as described previously (Thomas et al. 2001). Plasma membranes were stained with di-8-ANEPPS (10 µM, < 30 min), excited at 488 nm (emission > 505 nm). Double stranded DNA was stained with Hoescht 33258, excited at 364 nm (emission > 385 nm). For measurement of membrane potential (E_m), di-8-ANEPPS signals were background-subtracted, corrected for photobleach and expressed as f/f₀ ratios to normalise fluorescence levels (f) to starting fluorescence (f₀). Membrane capacitance was measured by integration or electronic compensation of the current transient following a small (-10 mV) voltage step.

Despite the morphological complexity of the demarcation membranes, the capacitative transient in > 95 % of MKs decayed with a single exponential and a typical initial series resistance (R_s) only 2-3 times (2.4 ± 1.0 MV; mean ± S.D., n = 68) higher than the pipette resistance (1.8 ± 0.5 MV). Di-8-ANEPPS, a styryl indicator with low membrane permeability extensively stained the volume of the MK, even between lobes of the polyploidic nucleus, as judged by co-staining with Hoescht 33258. In combined measurements of E_m and whole-cell voltage clamp with 70-75 % R_s compensation, voltage steps from -75 to 0 mV generated a uniform decrease in corrected f/f₀ 488 nm signals throughout the cell. This uniform voltage control, the single exponential decay of capacitative transients and the low series resistance in whole-cell recordings suggest that capacitance measurements represent a quantitative measurement of the entire DMS. MK capacitances covered more than a 10-fold range (64-694 pF) with an average of 236 ± 106 pF (n = 827). Based upon the capacitance of a single platelet of 128 fF (Maruyama, 1987), these MKs can generate 500-5000 platelets (average approx. 1850) if all surface-connected membrane in the precursor cell is used in the process of thrombopoiesis.

This work was funded by the British Heart Foundation and Medical Research Council.