Intracellular Ca2+ during metabolic activation of KATP channels in spontaneously active dorsal vagal neurons in medullary slices

Intracellular Ca2+ ([Ca2+](i)) and membrane properties were measured in fura-2 dialysed dorsal vagal neurons (DVN) spontaneously active at a frequency of 0.5-5 Hz. [Ca2+](i) increased by about 30 nM upon rising spike frequency by more than 200% due to 20-50 pA current pulses or 10 mu M serotonin. It fell by 30 nM upon block of spiking by current-injection, tetrodotoxin or Ni2+ and also during hyperpolarization due to gamma-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K+ (K-ATP) channels with diazoxide. KATP channel-mediated hyperpolarizations during anoxia or cyanide produced an initial [Ca2+](i) decrease which reversed into a secondary Ca2+ rise by less than 100 nM. Similar moderate rises of [Ca2+](i) were observed during block of aerobic metabolism under voltage-clamp as well as in intact cells, loaded with fura-2 AM. The magnitude of the metabolism-related [Ca2+](i) transients did not correlate with the amplitude of the KATP channel-mediated outward current. [Ca2+](i) did not change during diazoxide-induced or spontaneous activation of KATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing [Ca2+](i) with cyclopiazonic acid did not activate KATP channels. [Ca2+](i) was not affected upon block of outward current with sulphonylureas, but these KATP channel blockers were effective to reverse inhibition of spike discharge and, thus, the initial [Ca2+](i) fall upon spontaneous or diazoxide-, anoxia- and cyanide-induced K-ATP channel activation. A sulphonylurea-sensitive hyperpolarization and [Ca2+](i) fall was also revealed in the early phase of iodoacetate-induced metabolic arrest, whereas after about 20 min, occurrence of a progressive depolarization led to an irreversible rise of [Ca2+](i) to more than 1 mu M. The results indicate that K-ATP channel activity in DVN is not affected by physiological changes of intracellular Ca2+ and the lack of a major perturbance of Ca2+ homeostasis contributes to their high tolerance to anoxia.