Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro.

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Abstract
1. Simultaneous whole-cell patch-clamp and Fura-2 microfluorimetric recordings of calcium currents (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were made from neurones grown in primary culture from the dorsal root ganglion of the rat. 2. Cells held at -80 mV and depolarized to 0 mV elicited a ICa that resulted in an [Ca2+]i transient which was not significantly buffered during the voltage step and lasted long after the cell had repolarized and the current ceased. The process by which the cell buffered [Ca2+]i back to basal levels could best be described with a single-exponential equation. 3. The membrane potential versus ICa and [Ca2+]i relationship revealed that the peak of the [Ca2+]i transient evoked at a given test potential closely paralleled the magnitude of the ICa suggesting that neither voltage-dependent nor Ca2+-induced Ca2+ release from intracellular stores made a significant contribution to the [Ca2+]i transient. 4. When the cell was challenged with Ca2+ loads of different magnitude by varying the duration or potential of the test pulse, [Ca2+]i buffering was more effective for larger Ca2+ loads. The relationship between the integrated Ica and the peak of the [Ca2+]i transient reached an asymptote at large Ca2+ loads indicating that Ca2+-dependent processes became more efficient or that low-affinity processes had been recruited. 5. Inhibition of Ca2+ influx with neuropeptide Y demonstrated that inhibition of a large ICa produced minor alterations in the peak of the [Ca2+]i transient, while inhibition of smaller currents produced corresponding decreases in the [Ca2+]i transient. Thus, inhibition of the ICa was reflected by a change in the peak [Ca2+]i only when submaximal Ca2+ loads were applied to the cell, implying that modulation of [Ca2+]i is dependent on the activation state of the cells. 6. Intracellular dialysis with the mitochondrial Ca2+ uptake blocker Ruthenium Red in whole-cell patch-clamp experiments removed the buffering component which was responsible for the more efficient removal of [Ca2+]i observed when large Ca2+ loads were applied to the cell. 7. When cells were superfused with 50 mM-K+, [Ca2+]i transients recorded from the cell soma returned to control levels very slowly. Pharmacological studies indicated that mitochondria were cycling Ca2+ during this sustained elevation in [Ca2+]i. In contrast, [Ca2+]i transients recorded from cell processes returned to basal levels relatively rapidly. 8. Extracellular Na+-dependent Ca2+ efflx did not significantly contribute to buffering [Ca2+]i transients in dorsal root ganglion neurone cell bodies. Furthermore, calmodulin antagonists and sodium orthovanadate applied via the patch pipette were without effect on Ca2+ buffering. Thus, the Na+-Ca2+ exchange system does not participate in Ca2+ buffering in the cell bodies of dorsal root ganglion neurones and the ATP-dependent processes with presumably participate in removing Ca2+ from the cytosol do not seem to be regulated in the same manner as the Ca2+-Mg2+-ATPase previously characterized from erythrocytes.