Depolarization-evoked Ca2+ release in a non-excitable cell, the rat megakaryocyte

Abstract

1. The effect of membrane potential on [Ca2+]i in rat megakaryocytes was studied using simultaneous whole-cell patch clamp and fura-2 fluorescence recordings. 2. Depolarization from -75 to 0 mV had no effect on [Ca2+]i in unstimulated cells, but evoked one or more spikes of Ca2+ increase (peak increase: 714 +/- 95 nM) during activation of metabotropic purinoceptors by 1 microM ADP. 3. The depolarization-evoked Ca2+ increase was present in Ca2+-free medium and also following removal of Na+. Thus depolarization mobilizes Ca2+ from an intracellular store without a requirement for altered Na+-Ca2+ exchange activity. 4. Intracellular dialysis with heparin blocked the depolarization-evoked Ca2+ increase, indicating a role for functional IP3 receptors. 5. Under current clamp, ADP caused the membrane potential to fluctuate between -43 +/- 1 and -76 +/- 1 mV. Under voltage clamp, depolarization from -75 to -45 mV evoked a transient [Ca2+]i increase (398 +/- 91 nM) during exposure to ADP. 6. We conclude that during stimulation of metabotropic purinoceptors, membrane depolarization over the physiological range can stimulate Ca2+ release from intracellular stores in the rat megakaryocyte, a non-excitable cell type. This may represent an important mechanism by which electrogenic influences can control patterns of [Ca2+]i increase.

Figure 1. Depolarization-evoked [Ca²⁺]_i increases during exposure to ADP

A and B, simultaneous recordings of [Ca²⁺]_i and membrane potential (E_m) under whole-cell voltage clamp; the whole-cell current is also shown in A (middle trace). The bars indicate extracellular application of 1 μM ADP. A and B are from two different megakaryocytes representing the range of [Ca²⁺]_i responses to ADP and depolarization.

Figure 2. Depolarization-evoked [Ca²⁺]_i increase induced by ADP in Ca²⁺-free saline

Simultaneous recording of [Ca²⁺]_i and membrane potential in Ca²⁺-free saline with 0.5 mM EGTA. The cell was clamped at either -75 or 0 mV, except for two brief periods prior to the second step to 0 mV, when compensation for whole-cell capacitance and series resistance were checked using a 5 mV pulse at line frequency. The bar indicates application of 1 μM ADP.

Figure 3. Heparin blocks the [Ca²⁺]_i increase evoked by ADP and depolarization

Two sections of simultaneous [Ca²⁺]_i and membrane voltage recordings from a single voltage clamp experiment are shown, starting at 53 and 228 s after transition to the whole-cell configuration. The [Ca²⁺]_i axis range was limited in order to clearly illustrate the depolarization-evoked response. The pipette contained 10 mg ml⁻¹ heparin. The bars indicate application of 1 μM ADP.

Figure 4. Depolarization over the physiological range evokes an [Ca²⁺]_i increase

A, whole-cell current clamp recording of membrane potential during application of 1 μM ADP (bar). B, simultaneous recording of [Ca²⁺]_i and membrane potential during exposure to 1 μM ADP (bar) under whole-cell current clamp or voltage clamp as indicated by the double-ended arrows below. During voltage clamp, the cell was held at -75, -45 or 0 mV.