Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed

Abstract.

During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca²⁺, pulses of 100 µM ACh or 100 mM K⁺ of increasing duration (1–5 s) caused similar exocytosis of about 3–4 µC catecholamine. Depletion of endoplasmic reticulum (ER) Ca²⁺ by pretreatment with 1 µM thapsigargin, 10 mM caffeine and 10 µM ryanodine more than halved the responses to ACh but did not affect the responses to K⁺ responses. In these ER Ca²⁺-depleted cells the protonophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20 µM given during the 5 s preceding each pulse) augmented the responses to ACh responses fourfold for all pulse durations applied (1–5 s) whereas responses to K⁺ were potentiated twofold with 1 to 2 s pulses but were not affected with longer pulse durations. ACh pulses applied to fura-2-loaded cells evoked increases of bulk cytosolic [Ca²⁺] ([Ca²⁺]_c) that were substantially smaller than those elicited by K⁺ pulses. Confocal microscopy of fluo-3-loaded cells showed that ACh pulses elicited discrete and more localized [Ca²⁺]_c elevations, whereas K⁺ pulses produced higher [Ca²⁺]_c transients that spread out quickly throughout the cytosol. These results suggest that mitochondria sense the increase of local [Ca²⁺]_c elicited by ACh (that evokes firing of action potentials) much better than that induced by K⁺ (that produces sustained cell depolarisation). This implies that mitochondria are more sensitive to the local [Ca²⁺]_c changes resulting from the physiological triggering of action potentials by ACh.