Wanaverbecq, Nicolas Jean; (2002) Calcium homeostasis in rat sympathetic neurones: A primary role for the plasma membrane calcium ATPase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Patch-clamp experiments combined with indo-1 measurement of free intracellular Ca²⁺ concentration ([Ca²⁺]i) were used to determine the homeostatic systems involved in the maintenance of resting [Ca²⁺]i and the clearance of Ca²⁺ transients following activation of voltage-gated Ca²⁺ channels in neurones from rat superior cervical ganglion (SCG). In these neurones, the Ca²⁺ buffering capacity was estimated to be ≈ 1000 at [Ca²⁺]i close to rest and ≈ 250 at [Ca²⁺]i ≈ 1 μM and to involve at least two buffering systems with different affinities for Ca²⁺. Removal of extracellular Ca²⁺ led to a decrease in [Ca²⁺]i that was mimicked by the addition of La³⁺. This decrease in [Ca²⁺]i was more pronounced after inhibition of the endoplasmic reticulum Ca²⁺ uptake system (SERCA) and subsequent depletion of the intracellular stores. Inhibition of the plasma membrane Ca2+ pump (PMCA) by intracellular carboxyeosin or extracellular alkalisation (pH 9) both increased resting [Ca²⁺]i and prolonged the recovery of Ca²⁺ transients at peak [Ca²⁺]i < 500 nM. For [Ca²⁺]i loads > 500 nM, recovery showed an additional plateau phase that was abolished in carbonyl cyanide m-chlorohydrazone (CCCP) or on omitting Na⁺ from the intracellular solution. Inhibition of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) and of SERCA had a small but significant effect on the rate of decay of these larger Ca²⁺ transients. However, neither of these mechanisms appeared to contribute substantially to the recovery from rises in [Ca²⁺]i below 1 μM. In conclusion, resting [Ca²⁺]i is maintained as the result of a passive Ca²⁺ influx regulated by a large Ca²⁺ buffering system, Ca²⁺ extrusion via a PMCA and Ca²⁺ transport from the intracellular stores. PMCA is also the principal Ca²⁺ extrusion system at low Ca²⁺ loads, with additional participation of the NCX and intracellular organelles at high [Ca²⁺]i.

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