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 μm serotonin. It fell by 30 nm upon block of spiking by current-injection, tetrodotoxin or Ni²⁺ and also during hyperpolarization due to γ-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K⁺ (K_ATP) channels with diazoxide. K_ATP 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 K_ATP channel-mediated outward current. Ca²⁺]_i did not change during diazoxide-induced or spontaneous activation of K_ATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing Ca²⁺]_i with cyclopiazonic acid did not activate K_ATP channels. Ca²⁺]_i was not affected upon block of outward current with sulphonylureas, but these K_ATP 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 μm . 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.