Both myocardial ischaemia and anoxia cause massive noradrenaline release from cardiac sympathetic nerves. It has been hypothesised that this is mediated by leakage of noradrenaline from secretory vesicles and the activation of Na⁺-H⁺ exchange (NHE) leading to a rise in cytosolic Na⁺ and noradrenaline causing a reversal of Na⁺-noradrenaline co-transport (efflux) by uptake 1. The proposed role of NHE in this scheme is based upon the observation that amiloride analogues inhibit ischaemia- or anoxia-induced noradrenaline release (Kurz et al. 1996; Levi & Smith 2000). Surprisingly, there have been no direct studies of the effects of anoxia or ischaemia on NHE activity in sympathetic neurons; indeed there is little data on the role or function of NHEs in sympathetic neurons. Therefore, we have investigated the effect of anoxia on the regulation of intracellular pH (pH_i) in sympathetic neurons isolated from the middle cervical stellate ganglia (MCSG) of the rat. This ganglion complex represents the major source of postganglionic sympathetic nerves that supply the heart.

MCSG were excised from humanely killed 11- to 13-day-old Sprague-Dawley rat pups. Sympathetic neurons were disassociated from the ganglia by collagenase-trypsin digestion and were cultured under 5 % CO₂-air at 37 °C on glass coverslips coated with poly-L-lysine. Cells were used within 4 days and pH_i was determined using the pH indicators BCECF or SNARF-1.

The mean pH_i of single neurons was 7.07 ± 0.07 (± S.E.M., n = 76) when perfused with a Hepes buffered Tyrode solution (HBTS pH 7.4 at 37 °C). Under these conditions there was a rapid recovery of pH_i from an imposed acid load (NH₃/NH₄⁺ prepulse). Recovery from an acid load was prevented by the NHE inhibitor amiloride (1.5 mM) and under Na⁺-free conditions (NMDG substituted). Exposure to anoxic conditions (HBTS bubbled for >1 h with N₂ gas + 1 mM Na₂S₂O₄) caused a sustained decrease in pH_i within 10 min (ΔpH_i -0.23 ± 0.02 pH units, Student’s paired t test P < 0.001, n = 19). Under anoxic conditions there was substantially reduced pH_i recovery from an imposed acid load.

These data confirm the presence of NHE in sympathetic neurons and demonstrate that NHEs play a key role in pH_i regulation under normoxic conditions. However, during anoxia NHE activity appears to be markedly suppressed not activated. This observation is contrary to the hypothesis that neuronal NHE activity is increased during anoxia/ischaemia. The effects of other aspects of ischaemia upon neuronal NHE activity and the mechanisms of pH_i regulation in sympathetic neurons are currently under further investigation.

This work was funded by the Wellcome Trust.