Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed.
During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca2+, pulses of 100 microM ACh or 100 mM K+ of increasing duration (1-5 s) caused similar exocytosis of about 3-4 microC catecholamine. Depletion of endoplasmic reticulum (ER) Ca2+ by pretreatment with 1 microM thapsigargin, 10 mM caffeine and 10 microM 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 microM 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.
Cuchillo-Ibáñez
Inmaculada
I
Instituto Teófilo Hernando, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029, Spain.
Olivares
Román
R
Aldea
Marcos
M
Villarroya
Mercedes
M
Arroyo
Gloria
G
Fuentealba
Jorge
J
García
Antonio G
AG
Albillos
Almudena
A
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects*; D005456:Fluorescent Dyes; D016257:Fura-2; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D011188:Potassium / Q000494:pharmacology*; D013153:Splanchnic Nerves / Q000378:metabolism; D013569:Synapses / Q000502:physiology; D014475:Uncoupling Agents / Q000494:pharmacology
D002395:Catecholamines; D005456:Fluorescent Dyes; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium; D016257:Fura-2